U.S. patent application number 14/128447 was filed with the patent office on 2014-12-18 for methods of treating chronic disorders with complement inhibitors.
This patent application is currently assigned to Apellis Pharmaceuticals, Inc.. The applicant listed for this patent is Pascal Deschatelets, Cedric Francois. Invention is credited to Pascal Deschatelets, Cedric Francois.
Application Number | 20140371133 14/128447 |
Document ID | / |
Family ID | 47422981 |
Filed Date | 2014-12-18 |
United States Patent
Application |
20140371133 |
Kind Code |
A1 |
Francois; Cedric ; et
al. |
December 18, 2014 |
METHODS OF TREATING CHRONIC DISORDERS WITH COMPLEMENT
INHIBITORS
Abstract
In some aspects, the invention provides methods of treating a
subject in need of treatment for a chronic complement-mediated
disorder. In some aspects, the invention provides methods of
treating a subject in need of treatment for a Th17-associated
disorder. In some aspects, the invention provides methods of
treating a subject in need of treatment for a chronic respiratory
system disorder. In some aspects, the invention provides methods of
administering a complement inhibitor to a subject. In some
embodiments, a method of treating a subject comprises administering
multiple doses of a complement inhibitor to the subject according
to a dosing schedule that leverages the prolonged effect of
complement inhibition in chronic respiratory disorders. In some
embodiments, a subject has chronic obstructive pulmonary disease.
In some embodiments, a subject has asthma.
Inventors: |
Francois; Cedric;
(Louisville, KY) ; Deschatelets; Pascal;
(Louisville, KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Francois; Cedric
Deschatelets; Pascal |
Louisville
Louisville |
KY
KY |
US
US |
|
|
Assignee: |
Apellis Pharmaceuticals,
Inc.
Crestwood
KY
|
Family ID: |
47422981 |
Appl. No.: |
14/128447 |
Filed: |
June 22, 2012 |
PCT Filed: |
June 22, 2012 |
PCT NO: |
PCT/US2012/043845 |
371 Date: |
July 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61499895 |
Jun 22, 2011 |
|
|
|
Current U.S.
Class: |
514/1.7 ;
514/21.1 |
Current CPC
Class: |
G01N 33/6884 20130101;
A61B 5/14546 20130101; A61K 39/3955 20130101; A61P 27/02 20180101;
A61P 19/00 20180101; G01N 2800/12 20130101; A61P 27/00 20180101;
G01N 33/6869 20130101; A61B 5/414 20130101; A61P 21/04 20180101;
G01N 2800/14 20130101; A61P 11/00 20180101; A61P 21/00 20180101;
A61P 11/06 20180101; G01N 2800/122 20130101; A61K 38/10 20130101;
A61K 38/12 20130101; G01N 2800/24 20130101 |
Class at
Publication: |
514/1.7 ;
514/21.1 |
International
Class: |
A61K 38/12 20060101
A61K038/12; G01N 33/68 20060101 G01N033/68 |
Claims
1. A method of treating a subject in need of treatment for a
chronic respiratory disorder or other chronic complement-mediated
disorder, the method comprising administering multiple doses of a
complement inhibitor to the subject according to a dosing schedule
in which successive doses are administered on average (i) at least
2 weeks after the plasma concentration of the complement inhibitor
decreases to no more than 20% of the maximum plasma concentration
that was reached after the previous dose; (ii) at least 2 weeks
after plasma complement activation capacity has returned to at
least 50% of baseline after the previous dose; (iii) at intervals
equal to at least 2 times the terminal plasma half-life of the
complement inhibitor; or (iv) at intervals at least 3 weeks
apart.
2. The method of claim 1, wherein successive doses of the
complement inhibitor are administered on average (i) between 2
weeks and 6 weeks after the plasma concentration of the complement
inhibitor decreases to no more than 20% of the maximum plasma
concentration that was reached after the previous dose; (ii)
between 2 weeks and 6 weeks after plasma complement activation
capacity has returned to at least 50% of baseline after the
previous dose; (iii) at intervals equal to between 2 and 5 times
the terminal plasma half-life of the complement inhibitor; or (iv)
at intervals between 3 weeks and 6 weeks apart.
3. The method of claim 1, wherein successive doses of the
complement inhibitor are administered on average at least 4 weeks
apart.
4.-8. (canceled)
9. The method of claim 1, wherein at least 5 doses are
administered.
10. The method of claim 1, wherein the subject is in need of
treatment for asthma, chronic obstructive pulmonary disease (COPD),
or both.
11. (canceled)
12. The method of claim 1, wherein the complement inhibitor is
administered by the respiratory route.
13.-18. (canceled)
19. The method of claim 1, wherein the complement inhibitor
comprises an antibody, aptamer, peptide, polypeptide, or small
molecule that binds to C3, C5, factor B, or factor D.
20. The method of claim 1, wherein the complement inhibitor
comprises a compstatin analog.
21.-22. (canceled)
23. The method of claim 1, wherein the complement-mediated disorder
is a Th17-associated disorder.
24. The method of claim 1 comprising detecting a Th17 biomarker in
the subject or in a sample obtained from the subject.
25. The method of claim 24, wherein the Th17 biomarker is detected
in a sample comprising a body fluid, wherein the body fluid is
optionally selected from blood, BAL fluid, sputum, nasal secretion,
or urine or a combination thereof.
26. The method of claim 24, wherein the biomarker comprises at
least one cytokine that is produced by or promotes formation,
survival, or activity of Th17 cells.
27.-28. (canceled)
29. The method of claim 24, wherein the Th17 biomarker is detected
prior to administration of a dose of the complement inhibitor and
serves as an indicator that the subject is in need of a dose of the
complement inhibitor.
30. The method of claim 24, wherein the biomarker is detected prior
to administration of a dose of the complement inhibitor and serves
as an indicator that the subject is in need of a dose of the
complement inhibitor, and the method comprises administering the
complement inhibitor within a predetermined time period following
detection of the biomarker.
31. (canceled)
32. A method of treating a subject in need of treatment for a
chronic complement-mediated disorder, the method comprising: (a)
administering at least one dose of a complement inhibitor to the
subject; and (b) monitoring the subject for a Th17 biomarker in the
subject or in a sample obtained from the subject.
33.-35. (canceled)
36. The method of claim 32, wherein step (b) comprises detecting an
increased level of the biomarker as compared to a reference,
wherein the increased level indicates that the subject is in need
of a dose of the complement inhibitor, and the method further
comprises (c) administering at least one additional dose of the
complement inhibitor to the subject.
37.-58. (canceled)
59. A method of treating a subject having or at risk of a
Th17-associated disorder, the method comprising monitoring the
subject for evidence of a DC-Th17-B-Ab-C-DC cycle and administering
a complement inhibitor to the subject based at least in part on a
result of said monitoring.
60.-62. (canceled)
62. The method of claim 59, wherein the complement inhibitor
inhibits C3 activity or C3 activation.
63. (canceled)
64. The method of claim 59, wherein the anti-Th17 agent comprises
an antibody, small molecule, aptamer, or polypeptide that binds to
IL-1.beta., IL-6, IL-21, IL-17, or IL-23 or binds to receptor for
any of the foregoing.
65. The method of claim 59, wherein monitoring the subject for
evidence of a DC-Th17-B-Ab-C-DC cycle comprises assessing a
Th17-associated biomarker in the subject or in a sample obtained
from the subject.
66.-70. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a U.S. national phase application under
35. U.S.C. .sctn.371 of international PCT application no.
PCT/US2012/043845, filed Jun. 22, 2012, whe claims priority to U.S.
provisional patent application No. 61/499,895, filed Jun. 22, 2011.
The entire contents of these applications are hereby incorporated
by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 17, 2013, is named 2008575-0068_SL.txt and is 37,872 bytes
in size.
BACKGROUND OF THE INVENTION
[0003] Chronic disorders of the respiratory system are significant
causes of morbidity and mortality whose incidence is increasing
worldwide. According to World Health Organization estimates, about
80 million people have moderate to severe chronic obstructive lung
disease (COPD), and more than 3 million people died of COPD in 2005
(.about.5% of all deaths globally). COPD was the fifth leading
cause of death in 2002, and estimates suggest that it will be the
third leading cause of death worldwide in 2030 unless major risk
factors, particularly tobacco use, can be successfully curbed.
Asthma is also a significant global health problem, affecting an
estimated 300 million individuals worldwide. Both asthma and COPD
can have debilitating effects on patients' daily functioning and
quality of life, particularly when severe. These diseases also
represent significant burdens in terms of health care costs and
lost productivity.
[0004] Pharmacological therapies such as bronchodilators and
corticosteroids are widely used in the treatment of asthma and
COPD. However, a significant proportion of patients experience
persistent symptoms despite such interventions. Furthermore, these
agents can be associated with significant side effects. There is a
need for additional pharmacological therapies for treating
disorders affecting the respiratory system.
SUMMARY OF THE INVENTION
[0005] The invention provides, among other things, methods of
treating a chronic complement-mediated disorder, the methods
comprising administering a complement inhibitor to a subject in
need of treatment for the disorder. In some aspects, the invention
provides methods of treating a chronic disorder of the respiratory
system, the methods comprising administering a complement inhibitor
to a subject in need of treatment for the disorder. In some
embodiments, the disorder is asthma. In some embodiments, the
disorder is COPD. Certain aspects of the invention are based at
least in part on the recognition that complement inhibitors exhibit
a prolonged duration of effect in the treatment of chronic
complement-mediated disorders, e.g., chronic complement-mediated
disorders of the respiratory system, such as asthma or COPD. For
example, in some embodiments, the duration of action of a
complement inhibitor for significantly reducing one or more
manifestation(s) of a chronic complement-mediated disorder, e.g., a
chronic respiratory disorder, is greater than the duration of
action of the complement inhibitor for substantially inhibiting
plasma complement activation capacity when administered
intravenously.
[0006] In some aspects, the invention provides a method of treating
a subject in need of treatment for a chronic respiratory disorder
or other chronic complement-mediated disorder, the method
comprising administering multiple doses of a complement inhibitor
to the subject according to a dosing schedule in which successive
doses are administered on average (i) at least 2 weeks after the
plasma concentration of the complement inhibitor decreases to no
more than 20% of the maximum plasma concentration that was reached
after the previous dose; (ii) at least 2 weeks after plasma
complement activation capacity has returned to at least 50% of
baseline after the previous dose; (iii) at intervals equal to at
least 2 times the terminal plasma half-life of the complement
inhibitor; or (iv) at intervals at least 3 weeks apart. In some
embodiments successive doses of the complement inhibitor are
administered on average (i) between 2 weeks and 6 weeks after the
plasma concentration of the complement inhibitor decreases to no
more than 20% of the maximum plasma concentration that was reached
after the previous dose; (ii) between 2 weeks and 6 weeks after
plasma complement activation capacity has returned to at least 50%
of baseline after the previous dose; (iii) at intervals equal to
between 2 and 5 times the terminal plasma half-life of the
complement inhibitor; or (iv) at intervals between 3 weeks and 6
weeks apart. In some embodiments successive doses of the complement
inhibitor are administered on average at least 4 weeks apart. In
some embodiments successive doses of the complement inhibitor are
administered on average at least 2 weeks after plasma complement
activation capacity has returned to within the normal range after
the previous dose. In some embodiments successive doses of the
complement inhibitor are administered on average at least 2 weeks
after the plasma concentration of the complement inhibitor
decreases to no more than 10% of the maximum plasma concentration
that was reached after the previous dose. In some embodiments
successive doses of the complement inhibitor are administered on
average at least 2 weeks after the plasma concentration of the
complement inhibitor decreases to no more than 5% of the maximum
plasma concentration that was reached after the previous dose. In
some embodiments wherein the dosing schedule is determined based at
least in part on values of the complement inhibitor plasma
concentration, complement inhibitor plasma half-life, and/or plasma
complement activation capacity, as measured in a population of
subjects. In some embodiments the dosing schedule is determined
based at least in part on values of the complement inhibitor plasma
concentration, complement inhibitor plasma half-life, and/or plasma
complement activation capacity, of the subject being treated.
[0007] In some embodiments of any method comprising dosing, at
least 5 doses are administered.
[0008] In some embodiments a subject is in need of treatment for
asthma, chronic obstructive pulmonary disease (COPD), or both. In
some embodiments a subject is in need of treatment for severe
asthma.
[0009] In some embodiments a complement inhibitor is administered
by the respiratory route. In some embodiments a complement
inhibitor is administered using a nebulizer, metered dose inhaler,
or dry powder inhaler. In some embodiments a complement inhibitor
is administered using a vibrating mesh nebulizer.
[0010] In some embodiments a complement inhibitor is administered
by the intravenous route.
[0011] In some embodiments a complement inhibitor acts on C3 or
upstream of C3 in the complement cascade. In some embodiments the
complement inhibitor inhibits cleavage of C3, C5, or factor B.
[0012] In some embodiments a complement inhibitor comprises an
antibody, aptamer, peptide, polypeptide, or small molecule.
[0013] In some embodiments a complement inhibitor comprises an
antibody, aptamer, peptide, polypeptide, or small molecule that
binds to C3, C5, factor B, or factor D.
[0014] In some embodiments a complement inhibitor comprises a
compstatin analog.
[0015] In some embodiments a complement inhibitor comprises a
compstatin analog whose sequence comprises SEQ ID NO: 14, 21, 28,
29, 32, 33, 34, or 36.
[0016] In some embodiments a complement inhibitor comprises a
compstatin analog whose sequence comprises any of SEQ ID NOs:
3-41.
[0017] In some embodiments a complement-mediated disorder is a
Th17-associated disorder.
[0018] In some embodiments any method of treatment comprises
detecting a Th17 biomarker in the subject or in a sample obtained
from the subject. In some embodiments the Th17 biomarker is
detected in a sample comprising a body fluid, wherein the body
fluid is optionally selected from blood, BAL fluid, sputum, nasal
secretion, or urine or a combination thereof. In some embodiments
the biomarker comprises at least one cytokine that is produced by
or promotes formation, survival, or activity of Th17 cells. In some
embodiments an increased level of the Th17 biomarker as compared to
a reference indicates that the subject is in need of a dose of the
complement inhibitor. In some embodiments the reference is within
the normal range for persons not suffering from the disorder or is
a baseline value for the subject when the disorder is well
controlled. In some embodiments the Th17 biomarker is detected
prior to administration of a dose of the complement inhibitor and
serves as an indicator that the subject is in need of a dose of the
complement inhibitor. In some embodiments the biomarker is detected
prior to administration of a dose of the complement inhibitor and
serves as an indicator that the subject is in need of a dose of the
complement inhibitor, and the method comprises administering the
complement inhibitor within a predetermined time period following
detection of the biomarker. In some embodiments a predetermined
time period is 1, 2, 3, 4, 5, 6, or 7 days or 2, 3, or 4 weeks.
[0019] In some aspects, a method of treating a subject in need of
treatment for a chronic complement-mediated disorder comprises: (a)
administering at least one dose of a complement inhibitor to the
subject; and (b) monitoring the subject for a Th17 biomarker in the
subject or in a sample obtained from the subject. In some
embodiments the method, further comprises: (c) administering at
least one additional dose of the complement inhibitor to the
subject. In some embodiments step (b) comprises detecting a Th17
biomarker in the subject or in a sample obtained from the subject.
In some embodiments step (b) comprises detecting an increased level
of the biomarker as compared to a reference, wherein the increased
level indicates that the subject is in need of a dose of the
complement inhibitor. In some embodiments step (b) comprises
detecting an increased level of the biomarker as compared to a
reference, wherein the increased level indicates that the subject
is in need of a dose of the complement inhibitor, and the method
further comprises (c) administering at least one additional dose of
the complement inhibitor to the subject. In some embodiments step
(b) comprises detecting an increased level of the biomarker as
compared to a reference, wherein the increased level indicates that
the subject is in need of a dose of the complement inhibitor, and
the method further comprises (c) administering at least one
additional dose of the complement inhibitor to the subject within a
predetermined time of detecting the biomarker. In some embodiments
a predetermined time period is 1, 2, 3, 4, 5, 6, or 7 days or 2, 3,
or 4 weeks. In some embodiments a method further comprises
administering an anti-Th17 agent to the subject.
[0020] In some embodiments an anti-Th17 agent comprises an agent
that inhibits formation or activity of Th17 cells. In some
embodiments an anti-Th17 agent comprises an agent that inhibits the
production or activity of a cytokine produced by Th17 cells or that
promotes formation or activity of Th17 cells. In some embodiments
an anti-Th17 agent comprises an agent that inhibits the production
or activity of IL-1.beta., IL-6, IL-21, IL-22, IL-17, or IL-23. In
some embodiments an anti-Th17 agent comprises an antibody, small
molecule, aptamer, polypeptide, or RNAi agent. In some embodiments
an anti-Th17 agent comprises an antibody, small molecule, aptamer,
or polypeptide that binds to IL-1.beta., IL-6, IL-21, IL-22, IL-17,
or IL-23 or binds to receptor for any of the foregoing.
[0021] In some aspects, a pharmaceutical composition comprising a
complement inhibitor and an anti-Th17 agent is provided. In some
embodiments wherein the complement inhibitor inhibits C3 activity
or C3 activation. In some embodiments the complement inhibitor
comprises a compstatin analog. In some embodiments wherein the
anti-Th17 agent comprises an antibody, small molecule, aptamer, or
polypeptide that binds to IL-1.beta., IL-6, IL-21, IL-22, IL-17, or
IL-23 or binds to receptor for any of the foregoing.
[0022] In some aspects, a method of treating a complement-mediated
disorder comprises administering a composition comprising a
complement inhibitor and an anti-Th17 agent to a subject in need
thereo.
[0023] In some aspects, a method of treating a Th17-associated
disorder comprises administering a complement inhibitor and an
anti-Th17 agent to a subject in need thereof.
[0024] In some aspects, a method of method of disrupting a
DC-Th17-B-Ab-C-DCcycle is provided, the method comprising
administering comprising a complement inhibitor and an anti-Th17
agent to a subject in need thereof.
[0025] In some aspects, a method of treating a Th17-associated
disorder comprises administering a complement inhibitor and an
anti-Th17 agent to a subject in need thereof.
[0026] In some aspects, a method of treating a Th17-associated
disorder comprises administering a composition comprising a
complement inhibitor and an anti-Th17 agent to a subject in need
thereof.
[0027] In some aspects, a method of method of disrupting a
DC-Th17-B-Ab-C-DC cycle is provided, the method comprising
administering comprising a complement inhibitor and an anti-Th17
agent to a subject in need thereof.
[0028] In some embodiments, any of the methods comprises monitoring
the subject for evidence of a DC-Th17-B-Ab-C-DC cycle.
[0029] In some embodiments, any of the methods comprises monitoring
the subject for evidence of a DC-Th17-B-Ab-C cycle and
administering a complement inhibitor, anti-Th17 agent, or
composition comprising a complement inhibitor, anti-Th17 agent to
the subject based at least in part on a result of said
monitoring.
[0030] In some embodiments, any of the methods comprises monitoring
the subject for a Th17 biomarker.
[0031] In some embodiments, any of the methods comprises monitoring
the subject for a Th17 biomarker and administering a complement
inhibitor, anti-Th17 agent, or composition comprising a complement
inhibitor, anti-Th17 agent to the subject based at least in part on
a result of the monitoring.
[0032] In some aspects, a method of treating a subject having or at
risk of a complement-mediated disorder, comprises monitoring the
subject for evidence of a DC-Th17-B-Ab-C-DC cycle and administering
a complement inhibitor to the subject based at least in part on a
result of said monitoring. In some embodiments the method further
comprises administering an anti-Th17 agent to the subject.
[0033] In some aspects, a method of treating a subject having or at
risk of a complement-mediated disorder, comprises monitoring the
subject for evidence of a DC-Th17-B-Ab-C-DC cycle and administering
a complement inhibitor and an anti-Th17 agent to the subject based
at least in part on a result of said monitoring.
[0034] In some aspects, a method of treating a subject having or at
risk of a Th17-associated disorder, the method comprising
monitoring the subject for evidence of a DC-Th17-B-Ab-C-DC cycle
and administering a complement inhibitor to the subject based at
least in part on a result of said monitoring.
[0035] In some embodiments the method further comprises
administering an anti-Th17 agent to the subject.
[0036] In some aspects, a method of treating a subject having or at
risk of a Th17-associated disorder is provided, the method
comprising monitoring the subject for evidence of a
DC-Th17-B-Ab-C-DC cycle and administering a complement inhibitor
and an anti-Th17 agent to the subject based at least in part on a
result of said monitoring. In some embodiments the complement
inhibitor inhibits C3 activity or C3 activation. In some
embodiments the complement inhibitor comprises a compstatin
analog.
[0037] In some embodiments of a composition or method relating at
least in part to an anti-Th17 agent, the anti-Th17 agent comprises
an antibody, small molecule, aptamer, or polypeptide that binds to
IL-1.beta., IL-6, IL-21, IL-22, IL-17, or IL-23 or binds to
receptor for any of the foregoing.
[0038] In some embodiments of any method comprising monitoring a
subject for evidence of a DC-Th17-B-Ab-C-DC cycle, such monitoring
comprises assessing a Th17-associated biomarker in the subject or
in a sample obtained from the subject.
[0039] In some embodiments of any method comprising monitoring a
subject, the monitoring occurs approximately every 1-2 weeks, 2-4
weeks, or approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or
12 months.
[0040] In some embodiments of any method comprising administering a
complement inhibitor, anti-Th17 agent, or both, administration
occurs within no more than 1, 2, 3, 4, 5, 6, or 7 days or 2, 3, or
4 weeks of having detected evidence of a DC-Th17-B-Ab-C-DC cycle or
increased level of a Th17-associated biomarker.
[0041] In some aspects, a method of treating a subject in need of
treatment for AMD is provided, the method comprising administering
an anti-IL-23 agent to the subject. In some embodiments the agent
is administered locally to the eye, e.g., by intravitreal
injection. In some embodiments of the subject has dry AMD.
[0042] All articles, books, patent applications, patents, other
publications, websites, and databases mentioned in this application
are incorporated herein by reference. In the event of a conflict
between the specification and any of the incorporated references
the specification (including any amendments thereto) shall control.
Unless otherwise indicated, art-accepted meanings of terms and
abbreviations are used herein.
BRIEF DESCRIPTION OF THE DRAWING
[0043] FIGS. 1-11 are plots that show concentrations in
broncheoalveolar lavage (BAL) fluid of the indicated cytokines,
measured in samples obtained from individual cynomolgus monkeys at
the indicated time points prior to or following Ascaris suum
challenges 0, 1, and 2. Control animals (blue; triangles);
budesonide-treated animals (red; +); CA-28-treated animals (green;
circles). Plots of mean cytokine concentration at each time point
are superimposed and shown as continuous lines to more clearly
depict changes over the 24 hour time period.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
I. Definitions
[0044] As used herein, the term "antibody" encompasses antibodies
and antibody fragments comprising an antigen binding site.
Antibodies useful in certain embodiments of the invention could
originate from or be derived from various species, e.g., human,
non-human primate, rodent (e.g., mouse, rat, rabbit), goat,
chicken, and/or could be of various antibody classes, e.g., the
human classes: IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD,
and IgE. An antibody fragment (Fab) can be, for example, a Fab',
F(ab').sub.2, scFv (single-chain variable) or other fragment that
retains or contains an antigen binding site. See, e.g., Allen, T.,
Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references
therein. Antibodies known in the art as diabodies, minibodies, or
nanobodies can be used in various embodiments. Bispecific or
multispecific antibodies may be used in various embodiments. The
heavy and light chain of IgG immunoglobulins (e.g., rodent or human
IgGs) contain four framework regions (FR1 through FR4) separated
respectively by three complementarity determining regions (CDR1
through CDR3). The CDRs, particularly the CDR3 regions, especially
the heavy chain CDR3, are largely responsible for antibody
specificity. An antibody may be a chimeric antibody in which, for
example, a variable domain of rodent origin or non-human primate
origin is fused to a constant domain of human origin, or a
"humanized" antibody in which some or all of the
complementarity-determining region (CDR) amino acids that
constitute an antigen binding site (sometimes along with one or
more framework amino acids or regions) are "grafted" from a rodent
antibody (e.g., murine antibody) or phage display antibody to a
human antibody, thus retaining the specificity of the rodent or
phage display antibody. Thus, humanized antibodies may be
recombinant proteins in which only the antibody
complementarity-determining regions are of non-human origin. It
will be appreciated that the alterations to antibody sequence that
are involved in the humanization process are generally carried out
through techniques at the nucleic acid level, e.g., standard
recombinant nucleic acid techniques. In some embodiments only the
specificity determining residues (SDRs), the CDR residues that are
most crucial in the antibody-ligand interaction, are grafted. The
SDRs may be identified, e.g., through use of a database of the
three-dimensional structures of the antigen-antibody complexes of
known structures or by mutational analysis of the
antibody-combining site. In some embodiments an approach is used
that involves retention of more CDR residues, namely grafting of
so-called "abbreviated" CDRs, the stretches of CDR residues that
include all the SDRs. See, e.g., Kashmiri, S V, Methods.
36(1):25-34 (2005), for further discussion of SDR grafting. See,
e.g., Almagro J C, Fransson J. Humanization of antibodies. Front
Biosci. 13:1619-33 (2008) for review of various methods of
obtaining humanized antibodies. It will be understood that
"originate from or derived from" refers to the original source of
the genetic information specifying an antibody sequence or a
portion thereof, which may be different from the species in which
an antibody is initially synthesized. For example, "human" domains
may be generated in rodents whose genome incorporates human
immunoglobulin genes. See, e.g., Vaughan, et al, (1998), Nature
Biotechnology, 16: 535-539, e.g., to generate a fully human
antibody. An antibody may be polyclonal or monoclonal, though for
purposes of the present invention monoclonal antibodies are
generally preferred as therapeutic agents. Methods for generating
antibodies that specifically bind to virtually any molecule of
interest are known in the art. For example, monoclonal or
polyclonal antibodies can be purified from natural sources, e.g.,
from blood or ascites fluid of an animal that produces the antibody
(e.g., following immunization with the molecule or an antigenic
fragment thereof) or can be produced recombinantly, in cell culture
and, e.g., purified from culture medium. Affinity purification may
be used, e.g., protein A/G affinity purification and/or affinity
purification using the antigen as an affinity reagent. Suitable
antibodies can be identified using phage display and related
techniques. See, e.g., Kaser, M. and Howard, G., "Making and Using
Antibodies: A Practical Handbook" and Sidhu, S., "Phage Display in
Biotechnology and Drug Discovery", CRC Press, Taylor and Francis
Group, 2005, for further information. Methods for generating
antibody fragments are well known. For example, F(ab').sub.2
fragments can be generated, for example, through the use of an
Immunopure F(ab').sub.2 Preparation Kit (Pierce) in which the
antibodies are digested using immobilized pepsin and purified over
an immobilized Protein A column. The digestion conditions (such as
temperature and duration) may be optimized by one of ordinary skill
in the art to obtain a good yield of F(ab').sub.2. The yield of
F(ab').sub.2 resulting from the digestion can be monitored by
standard protein gel electrophoresis. F(ab') can be obtained by
papain digestion of antibodies, or by reducing the S--S bond in the
F(ab').sub.2. As used herein, a "single-chain Fv" or "scFv"
antibody fragment comprises the V.sub.H and V.sub.L domains of an
antibody, wherein these domains are present in a single polypeptide
chain. Typically, a scFv antibody further comprises a polypeptide
linker between the V.sub.H and V.sub.L domains, although other
linkers could be used to connect the domains in certain
embodiments.
[0045] The terms "approximately" or "about" in reference to a
number generally include numbers that fall within .+-.10%, in some
embodiments .+-.5%, in some embodiments .+-.1%, in some embodiments
.+-.0.5% of the number unless otherwise stated or otherwise evident
from the context (except where such number would impermissibly
exceed 100% of a possible value).
[0046] "Complement activation capacity" refers to the level of
complement activation that would result from exposure to a stimulus
that causes maximum complement activation. Typically, complement
activation capacity is assessed using a sample obtained from a
subject (e.g., a blood, plasma, serum, or other fluid sample, which
may be diluted appropriately), which sample may be exposed in vitro
to a complement activating stimulus. A heat-inactivated sample can
be used as a control. It will be understood that the stimulus need
not be sufficient to cause maximum complement activation in order
to provide a measurement of complement activation capacity. For
example, the extent to which complement activation occurs within a
defined time period can provide an indication of complement
activation capacity. Complement activation may be measured using,
e.g., a suitable assay such as a functional assay based on
hemolysis (e.g., lysis of sheep or chicken red blood cells);
deposition or capture of complement activation products (e.g., C3a,
C3b, iC3b, C5a, MAC), etc. Pathway-specific complement activation
capacity may be assessed using, e.g., appropriate stimuli and assay
conditions (e.g., presence or absence of calcium ions in the assay
composition) to activate one or more than one of the pathways. For
example, antibody (e.g., IgM or immune complexe) can be used to
activate the classical pathway; lipopolysaccharide (LPS) can be
used to activate the alternative pathway, mannan can beused to
activate the mannose-binding lectin portion of the lectin pathway,
etc. In some embodiments, the total classical complement activity
in a sample is measured using a CH50 test using antibody-sensitized
sheep or chicken erythrocytes as the activator of the classical
complement pathway and various dilutions of the test sample to
determine the amount required to give 50% lysis. The percent
hemolysis can be determined spectrophotometrically. The higher the
dilution of the sample that can still achieve 50% lysis (i.e., the
more diluted the sample), the greater complement activation
capacity. In some embodiments, an ELISA-based assay is used. In
some embodiments, complement activation is assessed based on iC3b
levels, e.g., substantially as described in PCT/US2010/035871
(WO2010135717) (see Examples). In some embodiments, complement
activation is assessed based on C3b levels, substantially as
described in PCT/US2008/001483 (WO/2008/097525) Examples 1 and 2,
respectively. In some embodiments, complement activation via the
classical pathway is assessed using the MicroVue CH50 Eq EIA Kit
(classical pathway), MicroVue Bb Plus EIA Kit (alternative
pathway), MicroVue iC3b EIA Kit, or MicroVue C3a Plus EIA Kit (all
from Quidel Corp.). In some embodiments, the amount of a complement
activation product is normalized to the amount of intact C3 present
in the sample prior to exposure to a complement activation
stimulus.
[0047] A "complement component" or "complement protein" is a
protein that is involved in activation of the complement system or
participates in one or more complement-mediated activities.
Components of the classical complement pathway include, e.g., C1q,
C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, and the C5b-9 complex,
also referred to as the membrane attack complex (MAC) and active
fragments or enzymatic cleavage products of any of the foregoing
(e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components of the
alternative pathway include, e.g., factors B, D, and properdin.
Components of the lectin pathway include, e.g., MBL2, MASP-1, and
MASP-2. Complement components also include cell-bound receptors for
soluble complement components, wherein such receptor mediates one
or more biological activities of such soluble complement component
following binding of the soluble complement component. Such
receptors include, e.g., C5a receptor (C5aR), C3a receptor (C3aR),
Complement Receptor 1 (CR1), Complement Receptor 2 (CR2),
Complement Receptor 3 (CR3, also known as CD45), etc. It will be
appreciated that the term "complement component" is not intended to
include those molecules and molecular structures that serve as
"triggers" for complement activation, e.g., antigen-antibody
complexes, foreign structures found on microbial or artificial
surfaces, etc.
[0048] A "complement regulatory protein" is a protein involved in
regulating complement activity. A complement regulatory protein may
down-regulate complement activity by, e.g., inhibiting complement
activation or by inactivating or accelerating decay of one or more
activated complement proteins. Examples of complement regulatory
proteins include C1 inhibitor, C4 binding protein, clusterin,
vitronectin, CFH, factor I, and the cell-bound proteins CD46, CD55,
CD59, CR1, CR2, and CR3.
[0049] "Linked", as used herein with respect to two or more
moieties, means that the moieities are physically associated or
connected with one another to form a molecular structure that is
sufficiently stable so that the moieties remain associated under
the conditions in which the linkage is formed and, preferably,
under the conditions in which the new molecular structure is used,
e.g., physiological conditions. In certain preferred embodiments of
the invention the linkage is a covalent linkage. In other
embodiments the linkage is noncovalent. Moieties may be linked
either directly or indirectly. When two moieties are directly
linked, they are either covalently bonded to one another or are in
sufficiently close proximity such that intermolecular forces
between the two moieties maintain their association. When two
moieties are indirectly linked, they are each linked either
covalently or noncovalently to a third moiety, which maintains the
association between the two moieties. In general, when two moieties
are referred to as being linked by a "linking moiety" or "linking
portion", the linkage between the two linked moieties is indirect,
and typically each of the linked moieties is covalently bonded to
the linking moiety. Two moieties may be linked using a "linker". A
linker can be any suitable moiety that reacts with the entities to
be linked within a reasonable period of time, under conditions
consistent with stability of the entities (portions of which may be
protected as appropriate, depending upon the conditions), and in
sufficient amount, to produce a reasonable yield. Typically the
linker will contain at least two functional groups, one of which
reacts with a first entity and the other of which reacts with a
second entity. It will be appreciated that after the linker has
reacted with the entities to be linked, the term "linker" may refer
to the part of the resulting structure that originated from the
linker, or at least the portion that does not include the reacted
functional groups. A linking moiety may comprise a portion that
does not participate in a bond with the entities being linked, and
whose main purpose may be to spatially separate the entities from
each other. Such portion may be referred to as a "spacer".
[0050] "Polypeptide", as used herein, refers to a polymer of amino
acids, optionally including one or more amino acid analogs. A
protein is a molecule composed of one or more polypeptides. A
peptide is a relatively short polypeptide, typically between about
2 and 60 amino acids in length, e.g., between 8 and 40 amino acids
in length. The terms "protein", "polypeptide", and "peptide" may be
used interchangeably. Polypeptides used herein may contain amino
acids such as those that are naturally found in proteins, amino
acids that are not naturally found in proteins, and/or amino acid
analogs that are not amino acids. As used herein, an "analog" of an
amino acid may be a different amino acid that structurally
resembles the amino acid or a compound other than an amino acid
that structurally resembles the amino acid. A large number of
art-recognized analogs of the 20 amino acids commonly found in
proteins (the "standard" amino acids) are known. One or more of the
amino acids in a polypeptide may be modified, for example, by the
addition of a chemical entity such as a carbohydrate group, a
phosphate group, a farnesyl group, an isofarnesyl group, a fatty
acid group, a linker for conjugation, functionalization, or other
modification, etc. Certain non-limiting suitable analogs and
modifications are described in WO2004026328 and/or below. The
polypeptide may be acetylated, e.g., at the N-terminus and/or
amidated, e.g., at the C-terminus.
[0051] In general, polypeptides may be obtained or produced using
any suitable method known in the art. For example, polypeptides may
be isolated from natural sources, produced in vitro or in vivo
using recombinant DNA technology in suitable expression systems
(e.g., by recombinant host cells or transgenic non-human animals or
plants), synthesized through chemical means such as solid phase
peptide synthesis and/or using methods involving chemical ligation
of synthesized peptides (see, e.g., Kent, S., J Pept Sci.,
9(9):574-93, 2003 and U.S. Pub. No. 20040115774), or a combination
of these. One of ordinary skill in the art would readily select
appropriate method(s). A polypeptide may comprise a tag, e.g., an
epitope tag, which tag may facilitate purification and/or detection
of the polypeptide. Exemplary tags include, e.g., 6Xhis (SEQ ID NO:
70), HA, Myc, SNUT, FLAG, TAP, etc. In some embodiments, a tag is
cleavable, e.g., the tag comprises a recognition site for cleavage
by a protease, or is separated from a portion complement inhibiting
portion of the polypeptide by a linking portion that comprises a
recognition site for cleavage by a protease. For example, a TEV
protease cleavage site can be used.
[0052] "Poxvirus" refers to a family of complex, double-stranded
DNA viruses constituting the family Poxyiridae. The family includes
the orthopoxviruses, a genus of the family Poxyiridae, subfamily
Chordopoxyirinae, comprising many species infecting mammals,
including human beings. Poxviruses are described in Fields, B N, et
al., Fields Virology, 3.sup.rd ed., Lippincott Williams &
Wilkins, 2001. Orthopoxviruses include, but are not limited to,
vaccinia virus, variola virus major, variola virus minor, cowpox
virus, monkeypox virus, camelpox virus, swinepox virus, and
ectromelia virus.
[0053] "Poxvirus complement control protein" refers to members of a
family of homologous proteins encoded by a number of different
poxviruses that bind to one or more complement pathway proteins and
inhibit either the classical pathway of complement activation, the
alternative pathway of complement activation, the lectin pathway,
or any combination of these. Poxvirus complement control proteins
are members of the complement control protein (CCP), also called
regulators of complement activation (RCA) superfamily (Reid, K B M
and Day, A J, Immunol Today, 10:177-80, 1989).
[0054] "Recombinant host cells", "host cells", and other such
terms, denote prokaryotic or eukaryotic cells or cell lines that
contain an exogenous nucleic acid (typically DNA) such as an
expression vector comprising a nucleic acid that encodes a
polypeptide of interest. It will be understood that such terms
include the descendants of the original cell(s) into which the
vector or other nucleic acid has been introduced. Appropriate host
cells include any of those routinely used in the art for expressing
polynucleotides (e.g., for purposes of producing polypeptide(s)
encoded by such polynucleotides) including, for example,
prokaryotes, such as E. coli; and eukaryotes, including for
example, fungi, such as yeast (e.g., Pichia pastoris); insect cells
(e.g., Sf9), plant cells, and animal cells, e.g., mammalian cells
such as CHO, R1.1, B-W, L-M, African Green Monkey Kidney cells
(e.g. COS-1, COS-7, BSC-1, BSC-40 and BMT-10) and cultured human
cells. The exogenous nucleic acid may be stably maintained as an
episome such as a plasmid or may at least in part be integrated
into the host cell's genome, optionally after being copied or
reverse transcribed. Terms such as "host cells", etc., are also
used to refer to cells or cell lines that can be used as recipients
for an exogenous nucleic acid, prior to introduction of the nucleic
acid. A "recombinant polynucleotide" is a polynucleotide that
contains nucleic acid sequences that are not found joined directly
to one another in nature. For example, the nucleic acid sequences
may occur in different genes or different species or one or more of
the sequence(s) may be a variant of a naturally occurring sequence
or may at least in part be an artificial sequence that is not
homologous to a naturally occurring sequence. A "recombinant
polypeptide" is a polypeptide that is produced by transcription and
translation of an exogenous nucleic acid by a recombinant host cell
or by a cell-free in vitro expression system and/or that contains
amino acid sequences that are not found joined directly to one
another in nature. In the latter case, the recombinant polypeptide
may be referred to as a "chimeric polypeptide". The amino acid
sequences in a chimeric polypeptide may, for example, occur in
different genes or in different species or one or more of the
sequence(s) may be a variant of a naturally occurring sequence or
may at least in part be an artificial sequence that is not
homologous to a naturally occurring sequence. It will be understood
that a chimeric polypeptide may comprise two or more polypeptide.
For example, first and second polypeptides A and B of a chimeric
polypeptide may be directly linked (A-B or B-A) or may be separated
by a third polypeptide portion C (A-C-B or B-C-A). In some
embodiments, portion C represents a polypeptide linker which may,
for example, comprise multiple glycine and/or serine residues. In
some embodiments, two or more polypeptides may be linked by
non-polypeptide linker(s).
[0055] "Reactive functional groups" as used herein refers to groups
including, but not limited to, olefins, acetylenes, alcohols,
phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic
acids, esters, amides, cyanates, isocyanates, thiocyanates,
isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo,
diazonium, nitro, nitriles, mercaptans, sulfides, disulfides,
sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals,
ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines,
imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic
acids thiohydroxamic acids, allenes, ortho esters, sulfites,
enamines, ynamines, ureas, pseudoureas, semicarbazides,
carbodiimides, carbamates, imines, azides, azo compounds, azoxy
compounds, and nitroso compounds, N-hydroxysuccinimide esters,
maleimides, sulfhydryls, and the like. Methods to prepare each of
these functional groups are well known in the art and their
application to or modification for a particular purpose is within
the ability of one of skill in the art (see, for example, Sandler
and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic
Press, San Diego, 1989, and Hermanson, G., Bioconjugate Techniques,
2.sup.nd ed., Academic Press, San Diego, 2008).
[0056] "Specific binding" generally refers to a physical
association between a target polypeptide (or, more generally, a
target molecule) and a binding molecule such as an antibody or
ligand. The association is typically dependent upon the presence of
a particular structural feature of the target such as an antigenic
determinant, epitope, binding pocket or cleft, recognized by the
binding molecule. For example, if an antibody is specific for
epitope A, the presence of a polypeptide containing epitope A or
the presence of free unlabeled A in a reaction containing both free
labeled A and the binding molecule that binds thereto, will reduce
the amount of labeled A that binds to the binding molecule. It is
to be understood that specificity need not be absolute but
generally refers to the context in which the binding occurs. For
example, it is well known in the art that numerous antibodies
cross-react with other epitopes in addition to those present in the
target molecule. Such cross-reactivity may be acceptable depending
upon the application for which the antibody is to be used. One of
ordinary skill in the art will be able to select antibodies or
ligands having a sufficient degree of specificity to perform
appropriately in any given application (e.g., for detection of a
target molecule, for therapeutic purposes, etc). It is also to be
understood that specificity may be evaluated in the context of
additional factors such as the affinity of the binding molecule for
the target versus the affinity of the binding molecule for other
targets, e.g., competitors. If a binding molecule exhibits a high
affinity for a target molecule that it is desired to detect and low
affinity for nontarget molecules, the antibody will likely be an
acceptable reagent. Once the specificity of a binding molecule is
established in one or more contexts, it may be employed in other,
preferably similar, contexts without necessarily re-evaluating its
specificity. In some embodiments, the affinity (as measured by the
equilibrium dissociation constant, Kd) of two molecules, e.g., two
molecules that exhibit specific binding, is 10.sup.-3 M or less,
e.g., 10.sup.-4 M or less, e.g., 10.sup.-5 M or less, e.g.,
10.sup.-6M or less, 10.sup.-7M or less, 10.sup.-8M or less, or
10.sup.-9M or less under the conditions tested, e.g., under
physiological conditions (e.g., conditions such as salt
concentration, pH, and/or temperature, etc., that reasonably
approximate corresponding conditions in vivo), or other conditions
of the assay. Binding affinity can be measured using any of a
variety of methods known in the art. For example, assays based on
isothermal titration calorimetry or surface plasmon resonance
(e.g., Biacore.RTM. assays) can be used in certain embodiments.
[0057] A "subject" treated according to the instant invention is
typically a human, a non-human primate, or another mammal (e.g., a
mouse or rat). It will be appreciated that, at least in embodiments
wherein a complement inhibitor is administered, the subject should
express at least one complement component that can be inhibited by
the particular complement inhibitor used. For example, a complement
inhibitor specific for primate complement would typically be
administered to a human or non-human primate or an animal model
that has been genetically engineered to express human complement
component(s). In some embodiments the subject is male. In some
embodiments the subject is female. In some embodiments, a human
subject is at least 12 years of age. In some embodiments a subject
is an adult, e.g., a human at least 18 years of age, e.g., between
18 and 100 years of age. In some embodiments a subject is at least
40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age. In some
embodiments the subject is a child, e.g., a human between 0 and 4
years of age, or between 5 and 11 years of age.
[0058] "Treating", as used herein in regard to treating a subject,
refers to providing treatment, i.e, providing any type of medical
or surgical management of a subject. The treatment can be provided
in order to reverse, alleviate, inhibit the progression of, prevent
or reduce the likelihood of a disease, or in order to reverse,
alleviate, inhibit or prevent the progression of, prevent or reduce
the likelihood of one or more symptoms or manifestations of a
disease. "Prevent" refers to causing a disease or symptom or
manifestation of a disease not to occur for at least a period of
time in at least some individuals, e.g., individuals at risk of
developing the disease, symptom, or manifestation. Treating can
include administering a compound or composition to the subject
following the development of one or more symptoms or manifestations
indicative of a disease, e.g., in order to reverse, alleviate,
reduce the severity of, and/or inhibit or prevent the progression
of the disease and/or to reverse, alleviate, reduce the severity
of, and/or inhibit or one or more symptoms or manifestations of the
disease. A compound or composition can be administered to a subject
who has developed a disease, or is at increased risk of developing
the disease relative to a member of the general population,
optionally a member who is matched with the subject in terms of
age, sex, and/or other demographic variable(s).
[0059] A "variant" of a particular polypeptide or polynucleotide
has one or more alterations (e.g., additions, substitutions, and/or
deletions, which may be referred to collectively as "mutations")
with respect to the polypeptide or nucleic acid, which may be
referred to as the "original polypeptide" or "original
polynucleotide", respectively. Thus a variant can be shorter or
longer than the polypeptide or polynucleotide of which it is a
variant. The terms "variant" encompasses "fragments". A "fragment"
is a continuous portion of a polypeptide that is shorter than the
original polypeptide. In certain embodiments of the invention a
variant polypeptide has significant sequence identity to the
original polypeptide over a continuous portion of the variant that
comprises at least 50%, preferably at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, or more, of the length of
the variant or the length of the polypeptide, (whichever is
shorter). In certain embodiments of the invention a variant
polypeptide has substantial sequence identity to the original
polypeptide over a continuous portion of the variant that comprises
at least 50%, preferably at least 60%, at least 70%, at least 80%,
at least 90%, at least 95%, or more, of the length of the variant
or the length of the polypeptide, (whichever is shorter). In a
non-limiting embodiment a variant has at least 80% identity to the
original sequence over a continuous portion of the variant that
comprises between 90% and 100% of the variant, e.g., over 100% of
the length of the variant or the length of the polypeptide,
(whichever is shorter). In another non-limiting embodiment a
variant has at least 80% identity to the original sequence over a
continuous portion of the variant that comprises between 90% and
100% of the variant, e.g., over 100% of the length of the variant
or the length of the polypeptide, (whichever is shorter). In
specific embodiments the sequence of a variant polypeptide has N
amino acid differences with respect to an original sequence,
wherein N is any integer between 1 and 10. In other specific
embodiments the sequence of a variant polypeptide has N amino acid
differences with respect to an original sequence, wherein N is any
integer between 1 and 20. An amino acid "difference" refers to a
substitution, insertion, or deletion of an amino acid.
[0060] In certain embodiments of the invention a fragment or
variant possesses sufficient structural similarity to the original
polypeptide so that when its 3-dimensional structure (either actual
or predicted structure) is superimposed on the structure of the
original polypeptide, the volume of overlap is at least 70%,
preferably at least 80%, more preferably at least 90% of the total
volume of the structure of the original polypeptide. A partial or
complete 3-dimensional structure of the fragment or variant may be
determined by crystallizing the protein, which can be done using
standard methods. Alternately, an NMR solution structure can be
generated, also using standard methods. A modeling program such as
MODELER (Sali, A. and Blundell, T L, J. Mol. Biol., 234, 779-815,
1993), or any other modeling program, can be used to generate a
predicted structure. If a structure or predicted structure of a
related polypeptide is available, the model can be based on that
structure. The PROSPECT-PSPP suite of programs can be used (Guo, J
T, et al., Nucleic Acids Res. 32(Web Server issue):W522-5, Jul. 1,
2004).
[0061] In many embodiments one, more than one, or all biological
functions or activities of a variant or fragment is substantially
similar to that of the corresponding biological function or
activity of the original molecule. In certain embodiments the
activity of a variant or fragment may be at least 20%, at least
50%, at least 60%, at least 70%, at least 80%, or at least 90% of
the activity of the original molecule, up to approximately 100%,
approximately 125%, or approximately 150% of the activity of the
original molecule. In certain embodiments an activity of a variant
or fragment is such that the amount or concentration of the variant
needed to produce an effect is within 0.5 to 5-fold of the amount
or concentration of the original molecule needed to produce that
effect. The invention contemplates use of variants of any of the
complement inhibiting polypeptides disclosed herein, wherein the
variant inhibits complement sufficiently to be useful in a method
described herein. In some embodiments, a variant lacks or has a
substantially reduction in a property that may be undesired such as
immunogenicity.
[0062] As used herein, "alkyl" refers to a saturated straight,
branched, or cyclic hydrocarbon having from about 1 to about 22
carbon atoms (and all combinations and subcombinations of ranges
and specific numbers of carbon atoms therein), with from about 1 to
about 12, or about 1 to about 7 carbon atoms being preferred in
certain embodiments of the invention. Alkyl groups include, but are
not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, t-butyl, n- pentyl, cyclopentyl, isopentyl, neopentyl,
n-hexyl, isohexyl, cyclohexyl, cyclooctyl, adamantyl,
3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.
[0063] As used herein, "halo" refers to F, Cl, Br or I.
[0064] As used herein, "alkanoyl" refers to an optionally
substituted straight or branched aliphatic acyclic residue having
about 1 to 10 carbon atoms (and all combinations and
subcombinations of ranges and specific number of carbon atoms)
therein, e.g., from about 1 to 7 carbon atoms which, as will be
appreciated, is attached to a terminal C.dbd.O group with a single
bond (and may also be referred to as an "acyl group"). Alkanoyl
groups include, but are not limited to, formyl, acetyl, propionyl,
butyryl, isobutyryl, pentanoyl, isopentanoyl, 2-methyl-butyryl,
2,2-dimethoxypropionyl, hexanoyl, heptanoyl, octanoyl, and the
like, and for purposes of the present invention a formyl group is
considered an alkanoyl group. "Lower alkanoyl" refers to an
optionally substituted straight or branched aliphatic acyclic
residue having about 1 to about 5 carbon atoms (and all
combinations and subcombinations of ranges and specific number of
carbon atoms). Such groups include, but are not limited to, formyl,
acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl,
etc.
[0065] As used herein, "aryl" refers to an optionally substituted,
mono- or bicyclic aromatic ring system having from about 5 to about
14 carbon atoms (and all combinations and subcombinations of ranges
and specific numbers of carbon atoms therein), with from about 6 to
about 10 carbons being preferred. Non-limiting examples include,
for example, phenyl and naphthyl.
[0066] As used herein, "aralkyl" refers to alkyl radicals bearing
an aryl substituent and having from about 6 to about 22 carbon
atoms (and all combinations and subcombinations of ranges and
specific numbers of carbon atoms therein), with from about 6 to
about 12 carbon atoms being preferred in certain embodiments.
Aralkyl groups can be optionally substituted. Non-limiting examples
include, for example, benzyl, naphthylmethyl, diphenylmethyl,
triphenylmethyl, phenylethyl, and diphenylethyl.
[0067] As used herein, the terms "alkoxy" and "alkoxyl" refer to an
optionally substituted alkyl-O-group wherein alkyl is as previously
defined. Exemplary alkoxy and alkoxyl groups include methoxy,
ethoxy, n-propoxy, i-propoxy, n-butoxy, and heptoxy.
[0068] As used herein, "carboxy" refers to a --C(.dbd.O)OH
group.
[0069] As used herein, "alkoxycarbonyl" refers to a
--C(.dbd.O)O-alkyl group, where alkyl is as previously defined.
[0070] As used herein, "aroyl" refers to a --C(.dbd.O)-aryl group,
wherein aryl is as previously defined. Exemplary aroyl groups
include benzoyl and naphthoyl.
[0071] The term "cyclic ring system" refers to an aromatic or
non-aromatic, partially unsaturated or fully saturated, 3- to
10-membered ring system, which includes single rings of 3 to 8
atoms in size and bi- and tri-cyclic ring systems which may include
aromatic 5- or 6-membered aryl or aromatic heterocyclic groups
fused to a non-aromatic ring. These heterocyclic rings include
those having from 1 to 3 heteroatoms independently selected from
the group consisting of oxygen, sulfur, and nitrogen. In certain
embodiments, the term heterocyclic refers to a non-aromatic 5-, 6-,
or 7-membered ring or a polycyclic group wherein at least one ring
atom is a heteroatom selected from the group consisting of O, S,
and N, including, but not limited to, a bi- or tri-cyclic group,
comprising fused six-membered rings having between one and three
heteroatoms independently selected from the group consisting of the
oxygen, sulfur, and nitrogen. In some embodiments, "cyclic ring
system" refers to a cycloalkyl group which, as used herein, refers
to groups having 3 to 10, e.g., 4 to 7 carbon atoms. Cycloalkyls
include, but are not limited to cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl and the like, which, is
optionally substituted. In some embodiments, "cyclic ring system"
refers to a cycloalkenyl or cycloalkynyl moiety, which is
optionally substituted.
[0072] Typically, substituted chemical moieties include one or more
substituents that replace hydrogen. Exemplary substituents include,
for example, halo, alkyl, cycloalkyl, aralkyl, aryl, sulfhydryl,
hydroxyl (--OH), alkoxyl, cyano (--CN), carboxyl (--COOH),
--C(.dbd.O)O-alkyl, aminocarbonyl (--C(.dbd.O)NH.sub.2),
--N-substituted aminocarbonyl (--C(.dbd.O)NHR''), CF.sub.3,
CF.sub.2CF.sub.3, and the like. In relation to the aforementioned
substituents, each moiety R'' can be, independently, any of H,
alkyl, cycloalkyl, aryl, or aralkyl, for example.
[0073] As used herein, "L-amino acid" refers to any of the
naturally occurring levorotatory alpha-amino acids normally present
in proteins or the alkyl esters of those alpha-amino acids. The
term "D-amino acid" refers to dextrorotatory alpha-amino acids.
Unless specified otherwise, all amino acids referred to herein are
L-amino acids.
[0074] As used herein, an "aromatic amino acid" is an amino acid
that comprises at least one aromatic ring, e.g., it comprises an
aryl group.
[0075] As used herein, an "aromatic amino acid analog" is an amino
acid analog that comprises at least one aromatic ring, e.g., it
comprises an aryl group.
II. Methods of Treating Disorders Using Complement Inhibitors
[0076] The present invention provides, among other things, methods
of treating chronic complement-mediated disorders using complement
inhibitors. For example, the invention provides methods of treating
chronic respiratory system disorders using complement inhibitors.
In some aspects, the inventive methods are based at least in part
on the recognition that complement inhibitors have a prolonged
duration of action in treating a variety of disorders, e.g.,
chronic respiratory disorders, as compared, for example, with their
plasma half-life and/or their duration of action for inhibiting
plasma complement activation capacity. In some aspects, the
invention provides methods of treating a chronic
complement-mediated disorder by administering multiple doses of a
complement inhibitor, wherein the complement inhibitor is
administered according to a dosing schedule that utilizes the
prolonged effect of complement inhibition.
[0077] As used herein, a "chronic disorder" is a disorder that
persists for at least 3 months and/or is accepted in the art as
being a chronic disorder. In many embodiments, a chronic disorder
persists for at least 6 months, e.g., at least 1 year, or more,
e.g., indefinitely. One of ordinary skill in the art will
appreciate that at least some manifestations of various chronic
disorders may be intermittent and/or may wax and wane in severity
over time. A chronic disorder may be progressive, e.g., having a
tendency to become more severe or affect larger areas over time. A
number of chronic complement-mediated disorders are discussed
herein. Varioue embodiments of the invention pertaining to chronic,
complement-mediated respiratory disorders, in particular asthma and
COPD, are discussed in most detail herein, but it should be
understood that the various aspects of the invention encompass
embodiments pertaining to any chronic complement-mediated disorder
including, but not limited to, the specific disorders disclosed
herein. Accordingly, where an embodiment herein refers to a chronic
respiratory disorder, the invention provides analogous embodiments
pertaining to other complement-mediated disorders, e.g., chronic
disorders in which complement activation (e.g., excessive or
inappropriate complement activation) is involved, e.g., as a
contributing and/or at least partially causative factor. For
convenience, disorders are sometimes grouped by reference to an
organ or system that is often particularly affected in subjects
suffering from the disorder. It will be appreciated that a number
of disorders can affect multiple organs or systems, and the
classification herein is in no way limiting. Furthermore, a number
of manifestations (e.g., symptoms) may occur in subjects suffering
from any of a number of different disorders. In some aspects, the
invention provides methods of treating a subject in need of
treatment for such manifestation(s), e.g., methods for alleviating
such manifestation(s), the methods comprising administering a
complement inhibitor to the subject according to an inventive
dosing schedule (e.g., a dosing schedule that employs an inventive
dosing interval). In some embodiments, a subject suffers from
multiple complement-mediated disorders. Non-limiting information
regarding disorders of interest herein may be found, e.g., in
standard textbooks of internal medicine such as Cecil Textbook of
Medicine (e.g., 23rd edition), Harrison's Principles of Internal
Medicine (e.g., 17th edition), and/or standard textbooks focusing
on particular areas of medicine, particular body systems or organs,
and/or particular disorders.
[0078] In some embodiments, a chronic complement-mediated disorder
is a Th2-associated disorder. As used herein, a Th2-associated
disorder is a disorder characterized by an excessive number and/or
excessive or inappropriate activity of CD4+ helper T cells of the
Th2 subtype ("Th2 cells") in the body or a portion thereof, e.g.,
in at least one tissue, organ, or structure. For example, there may
be a predominance of Th2 cells relative to CD4+ helper T cells of
the Th1 subtype ("Th1 cells") e.g., in at least one tissue, organ,
or structure affected by a disorder. As known in the art, Th2 cells
typically secrete characteristic cytokines such as interleukin-4
(IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13), while Th1
cells typically secrete interferon-.gamma. (IFN-.gamma.) and tumor
necrosis factor .beta. (TNF .beta.). In some embodiments, a
Th2-associated disorder is characterized by excessive production
and/or amount of IL-4, IL-5, and/or IL-13, e.g., relative to
IFN-.gamma. and/or TNF .beta. e.g., in at least some at least one
tissue, organ, or structure.
[0079] In some embodiments, a chronic complement-mediated disorder
is a Th17-associated disorder. As used herein, a Th17-associated
disorder is a disorder characterized by an excessive number and/or
excessive or inappropriate activity of CD4+ helper T cells of the
Th17 subtype ("Th17 cells") in the body or a portion thereof, e.g.,
in at least one tissue, organ, or structure. For example, there may
be a predominance of Th17 cells relative to Th1 and/or Th2 cells,
e.g., in at least one tissue, organ, or structure affected by a
disorder. In some embodiments a predominance of Th17 cells is a
relative predominance, e.g., the ratio of Th17 cells to Th1 cells
and/or the ratio of Th17 cells to Th2 cells, is increased relative
to normal values. In some embodiments the ratio of Th17 cells to T
regulatory cells (CD4.sup.+CD25.sup.+ regulatory T cells, also
termed "Treg cells"), is increased relative to normal values.
Formation of Th17 cells and/or activation of Th 17 cells is
promoted by various cytokines, e.g., interleukin 6 (IL-6),
interleukin 21 (IL-21), interleukin 23 (IL-23), and/or interleukin
1.beta. (IL-1.beta.). Formation of Th17 cells encompasses
differentiation of precursor T cells, e.g., naive CD4+ T cells,
towards a Th17 phenotype and their maturation into functional Th17
cells. In some embodiments, formation of Th17 cells encompasses any
aspect of development, proliferation (expansion), survival, and/or
maturation of Th17 cells. In some embodiments, a Th17-associated
disorder is characterized by excessive production and/or amount of
IL-6, IL-21, IL-23, and/or IL-1.beta.. Th17 cells typically secrete
characteristic cytokines such as interleukin-17A (IL-17A),
interleukin-17F (IL-17F), interleukin-21 (IL-21), and
interleukin-22 (IL-22). In some embodiments, a Th17-associated
disorder is characterized by excessive production and/or amount of
a Th17 effector cytokine, e.g., IL-17A, IL-17F, IL-21, and/or
IL-22. In some embodiments excessive production or amount of a
cytokine is detectable in the blood. In some embodiments excessive
production or amount of a cytokine is detectable locally, e.g., in
at least one tissue, organ or structure. In some embodiments a
Th17-associated disorder is associated with a decreased number of
Tregs and/or decreased amount of a Treg-associated cytokine. In
some embodiments a Th17 disorder is any chronic inflammatory
disease, which term encompasses a range of ailments characterized
by self-perpetuating immune insults to a variety of tissues and
that seem to be dissociated from the initial insult that caused the
ailment (which may be unknown). In some embodiments a
Th17-associated disorder is any autoimmune disease. Many if not
most "chronic inflammatory diseases" may in fact be auto-immune
diseases. Examples of Th17-associated disorders include
inflammatory skin diseases such as psoriasis and atopic dermatitis;
systemic scleroderma and sclerosis; inflammatory bowel disease
(IBD) (such as Crohn's disease and ulcerative colitis); Behcet's
Disease; dermatomyositis; polymyositis; multiple sclerosis (MS);
dermatitis; meningitis; encephalitis; uveitis; osteoarthritis;
lupus nephritis; rheumatoid arthritis (RA), Sjorgen's syndrome,
multiple sclerosis, vasculitis; central nervous system (CNS)
inflammatory disorders, chronic hepatitis; chronic pancreatitis,
glomerulonephritis; sarcoidosis; thyroiditis, pathologic immune
responses to tissue/organ transplantation (e.g., transplant
rejection); COPD, asthma, bronchiolitis, hypersensitivity
pneumonitis, idiopathic pulmonary fibrosis (IPF), periodontitis,
and gingivitis. In some embodiments a Th17 disease is a classically
known auto-immmune disease such as Type I diabetes or psoriasis. In
some embodiments a Th17-associated disorder is age-related macular
degeneration.
[0080] In some aspects, the present disclosure provides the insight
that complement activation and Th17 cells participate in a cycle
that involves dendritic cells and antibodies and that contributes
to maintenance of a pathologic immunologic microenvironment
underlying a range of disorders. Without wishing to be bound by any
theory, the pathologic immunologic microenvironment, once
established, is self-sustaining and contributes to cell and tissue
injury. Dendritic cells (DCs) are a type of white blood cell that
occur in most tissues of the body, particularly those exposed to
the external environment, such as skin and mucosal surfaces, and in
the blood (where they may be found in an immature state). Immature
DCs sample the surrounding environment for pathogens through, e.g.,
pattern recognition receptors such as toll-like receptors (TLRs).
In response to various stimuli (e.g., pathogen-associated
substances or other danger signals, inflammatory cytokines, and/or
antigen-activated T cells), DCs mature and migrate to lymphoid
tissues, where they act as antigen-presenting cells and activate
other immune system cells, such as T cells and B cells, by
presenting them with antigen fragments together with non-antigen
specific costimulatory molecules. DC stimulation promotes Th cell
proliferation, activation, and differentiation into effector Th
cells. Effector Th cells "help" cytotoxic T cells, B cells, and
macrophages by, e.g., secreting cytokines that have various
stimulatory effects. Th help can, for example, enhance
proliferation and activation of cytotoxic T cells, stimulate B cell
proliferation and maturation and antibody production. Of particular
importance in accordance with certain aspects of the present
disclosure, mature DCs are capable of causing CD4+ helper T cells
to differentiate into Th17 cells, which in turn stimulate
maturation and activation of B cells, resulting in production of
antibodies.
[0081] The antibody response is generally polyclonal, with most
antibodies being of low affinity. However, certain of these
antibodies may be cross-reactive with self proteins, such as self
proteins that have been enzymatically or non-enzymatically
chemically modified in the body post-translationally in any of a
variety of ways. Such self proteins may, for example, be exposed at
the surface of cells, present in the interstitial space, and/or
circulating in the blood. Modifications of self proteins may
include, e.g., acylation and/or glycation (non-enzymatic formation
of a covalent bond between a protein or lipid and a sugar). For
example, proteins can be oxidized in numerous ways, which can be
classified into at least three categories. A first mechanism
involves oxidative cleavages in either the protein backbone or
amino acid side chains, e.g., side chains of Pro, Arg, Lys, Thr,
Glu or Asp residues, which may occur by direct oxidation with
reactive oxygen species (ROS). Certain ROS are produced during
normal cellular metabolism, and various mechanisms exist to defend
against the potentially damaging effects of such compounds.
Examples of ROS include, e.g., superoxide anion, hydrogen peroxide,
and peroxynitrite. Excessive levels of reactive oxygen species
(ROS) can result from the environment and/or defects in cellular
processes or antioxidant mechanisms, resulting in high levels of
oxidative stress. A second mechanism of protein oxidation is by
addition of lipid oxidation products such as 4-hydroxy-2-noneal,
2-propenal or malondialdehyde to proteins. In a third mechanism,
carbonyl groups are generated in proteins by oxidation of advanced
glycation end (AGE) products. AGEs can form as a result of a chain
of chemical reactions after an initial glycation reaction. Examples
of AGE-modified sites are carboxymethyllysine (CML) and
carboxyethyllysine (CEL). ROS can degrade polyunsaturated lipids,
forming malondialdehyde, a reactive aldehyde that forms covalent
protein adducts referred to as advanced lipoxidation end-products
(ALEs). Carboxyethylpyrrole (CEP) protein modifications are
generated from oxidation of docosahexaenoate-containing lipids.
[0082] Modified self proteins (e.g., malondialdehyde-modified
proteins, CEP-modified proteins) may contain epitopes recognized as
non-self by the immune system, e.g., by antibodies. Binding of
antibodies to self proteins leads to complement activation, e.g.,
via the classical pathway. Once initiated, classical
pathway-mediated complement activation is amplified by the
alternative pathway. In accordance with certain aspects of the
present disclosure, activated complement polarizes DCs to sustain
the Th17 phenotype. For example, DCs may be polarized towards
secretion of cytokines such as IL-23 that promote Th17 formation
and/or activation. Complement cleavage products such as the
anaphylotoxins (e.g., C3a, C4a, and/or C5a) and/or products of C3
cleavage and degradation such as iC3b or C3d may bind to DC cell
surface receptors and contribute towards polarizing DCs to sustain
the Th17 phenotype. An example of how complement can polarize DCs
is the activation of dendritic cells by aluminum oxide. Aluminum
oxide is widely used as an adjuvant to vaccines. Aluminum oxide
activates complement and this stimulates DCs into promoting and
sustaining Th2 and Th17 phenotypes. Complement can polarize other
types of antigen-presenting cells as well. Monocytes and
macrophages can act as antigen-presenting cells and can similarly
be polarized by complement activation. In some aspects, the cycle
may be summarized as follows: (1) Mature dendritic cells in an
environment of high complement activation stimulate Th17 cell
phenotypic differentiation; (2) Th17 T cells stimulate polyclonal
B-cell expansion, leading to the production of polyclonal,
self-reactive antibodies against, e.g., modified self proteins,
such as carbonyl-modified self-proteins; (3) Carbonyl-modified
self-proteins can be generated as a result of oxidative stress.
This can arise, for example, from pollutants, cigarette smoke, or
allergens; (4) Self-reactive antibodies against carbonyl-modified
self-proteins help promote or sustain an environment of high
complement activation; (5) High complement activation drives
antigen-presenting cells into sustaining a Th17
micro-environment.
[0083] The effector pathways that lead this cycle to inflict tissue
damage can be varied, but, without wishing to be bound by any
theory, it is believed that a principal pathway is via macrophages.
In some aspects, IL-17 secreted by Th17 cells, itself or in
combination with one or more other cytokines such as interferon
gamma (IFN-.gamma.) contributes to macrophage activation and/or
polarization towards an M1 phenotype. M1-polarized macrophages are
immune effector cells that are characterized by expression of high
levels of proinflammatory cytokines, high production of reactive
nitrogen and oxygen intermediates, and may exhibit strong cytotoxic
activity against targets such as microbes and tumor cells.
Macrophages, e.g., M1-polarized macrophages, and the products they
produce can lead to tissue damage and are important mediators of
immunopathology. Modification of self proteins and other cellular
components by reactive nitrogen and oxygen species can render them
dysfunctional, thereby interfering with normal cellular processes.
Dysfunctional modified proteins can accumulate to toxic levels,
which can lead to cell death. Macrophages are also capable of
direct killing of altered self cells, e.g., self cells that have
oxidatively modified proteins or lipids exposed at their cell
surface. Reactive nitrogen and oxygen species produced by
macrophages can amplify oxidative stress, resulting in further
modification of self proteins by mechanisms such as those described
above, which produces new targets for self-reactive antibodies and
macrophages. The antibodies further activate complement, which
maintains DC polarization towards a Th17-promoting phenotype. Thus,
a vicious cycle is perpetuated in which Th17 cells activate B
cells, resulting in polyclonal antibody production and consequent
complement activation, which in term promotes DC polarization
towards a Th17-promoting phenotype that drives continued
stimulation of B cells and antibody production. For purposes
hereof, this cycle, also summarized above, may be referred to as
the "dendritic cell--Th 17 cell--B
cell-antibody-complement-dendritic cell" cycle, abbreviated as
DC-Th17-B-Ab-C-DC cycle. Polarization of macrophages to an M1
phenotype and production of ROS that can directly damage cellular
components may occur as "outputs" of this feedback loop. The
pathologic consequences that result from DC-Th17-B-Ab-C-DC cycle
and its outputs may vary in different tissues or organs. For
example, in the respiratory system, they may at least in part
underlie chronic respiratory diseases such as asthma and COPD. In
the eye, they may at least in part underlie chronic disorders such
as age-related macular degeneration. In the skin, they may at least
in part underlie psoriasis. In the pancreas, they may at least in
part underlie Type I diabetes.
[0084] In some embodiments, a chronic complement-mediated disorder
is an IgE-associated disorder. As used herein, an "IgE-associated
disorder" is a disorder characterized by excessive and/or
inappropriate production and/or amount of IgE, excessive or
inappropriate activity of IgE producing cells (e.g., IgE producing
B cells or plasma cells), and/or excessive and/or inappropriate
activity of IgE responsive cells such as eosinophils or mast cells.
In some embodiments, an IgE-associated disorder is characterized by
elevated levels of total IgE and/or in some embodiments,
allergen-specific IgE, in the plasma of a subject and/or
locally.
[0085] In some embodiments, a chronic complement-mediated disorder
is characterized by complement-mediated hemolysis, e.g.,
complement-mediated hemolysis attributable to deficiency or
mutation of one or more endogenous complement regulatory proteins.
In some embodiments, a chronic complement-mediated disorder is not
characterized by hemolysis attributable, e.g., to deficiency or
mutation of one or more endogenous complement regulatory
proteins.
[0086] In some embodiments, a chronic complement-mediated disorder
is characterized by the presence of autoantibodies and/or immune
complexes in the body, which may activate complement via, e.g., the
classical pathway. Autoantibodies may, for example, bind to self
antigens, e.g., on cells or tissues in the body. In some
embodiments, autoantibodies bind to antigens in blood vessels,
skin, nerves, muscle, connective tissue, heart, kidney, thyroid,
etc. In some embodiments, a chronic complement-mediated disorder is
not characterized by autoantibodies and/or immune complexes.
[0087] In some embodiments, the invention provides methods for
treating a chronic complement-mediated disorder by administering
multiple doses of a complement inhibitor, wherein the complement
inhibitor is administered according to a dosing schedule that
utilizes the prolonged effect of complement inhibition. "Dosing
schedule" refers to the timing of administration of a compound (or
composition containing a compound). In some embodiments, an
inventive method utilizes an increased dosing interval as compared,
for example, with a dosing interval that aims to maintain a
significant level of complement inhibitor and/or a significant
level of complement inhibition in the body substantially throughout
a treatment period. In some embodiments, an inventive method
utilizes an increased dosing interval as compared, for example,
with a dosing interval that aims to expose tissue(s) or organ(s)
affected by a complement-mediated disorder to a significant level
of complement inhibitor and/or maintain a significant level of
complement inhibition in such tissue(s) or organ(s) (and/or in body
fluids contacting or within such tissue(s) or organ(s))
substantially throughout a treatment period. As used herein,
"dosing interval" refers to the time interval between
administration of successive doses of a compound (or composition
comprising a compound).
[0088] In some embodiments, a chronic complement-mediated disorder
is a respiratory disorder. In some embodiments, a chronic
respiratory disorder is asthma or chronic obstructive pulmonary
disease (COPD). In some embodiments, a chronic respiratory disorder
is pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis),
radiation-induced lung injury, allergic bronchopulmonary
aspergillosis, hypersensitivity pneumonitis (also known as allergic
alveolitis), eosinophilic pneumonia, interstitial pneumonia,
sarcoid, Wegener's granulomatosis, or bronchiolitis obliterans.
[0089] In some embodiments, a chronic complement-mediated disorder
is allergic rhinitis, rhinosinusitis, or nasal polyposis. In some
embodiments, the invention provides a method of treating a subject
in need of treatment for allergic rhinitis, rhinosinusitis, or
nasal polyposis, the method comprising administering a complement
inhibitor according to a dosing schedule described herein to a
subject in need of treatment for the disorder.
[0090] In some embodiments, a chronic complement-mediated disorder
is a disorder that affects the musculoskeletal system. Examples of
such disorders include inflammatory joint conditions (e.g.,
arthritis such as rheumatoid arthritis or psoriatic arthritis,
juvenile chronic arthritis, spondyloarthropathies Reiter's
syndrome, gout). In some embodiments, a musculoskeletal system
disorder results in symptoms such as pain, stiffness and/or
limitation of motion of the affected body part(s). Inflammatory
myopathies include dermatomyositis, polymyositis, and various
others are disorders of chronic muscle inflammation of unknown
etiology that result in muscle weakness. In some embodiments, a
chronic complement-mediated disorder is myasthenia gravis. In some
embodiments, the invention provides a method of treating any of the
foregoing disorders affecting the musculoskeletal system, the
method comprising administering a complement inhibitor according to
a dosing schedule described herein to a subject in need of
treatment for the disorder.
[0091] In some embodiments, a chronic complement-mediated disorder
is a disorder that affects the integumentary system. Examples of
such disorders include, e.g., atopic dermatitis, psoriasis,
pemphigus, systemic lupus erythematosus, dermatomyositis,
scleroderma, sclerodermatomyositis, Sjogren syndrome, and chronic
urticaria. In some aspects, the invention provides a method of
treating any of the foregoing disorders affecting the integumentary
system, the method comprising administering a complement inhibitor
according to a dosing schedule described herein to a subject in
need of treatment for the disorder.
[0092] In some embodiments, a chronic complement-mediated disorder
affects the nervous system, e.g., the central nervous system (CNS)
and/or peripheral nervous system (PNS). Examples of such disorders
include, e.g., multiple sclerosis, other chronic demyelinating
diseases, amyotrophic lateral sclerosis, chronic pain, stroke,
allergic neuritis, Huntington's disease, Alzheimer's disease, and
Parkinson's disease. In some embodiments, the invention provides a
method of treating any of the foregoing disorders affecting the
nervous system, the method comprising administering a complement
inhibitor according to a dosing schedule described herein to a
subject in need of treatment for the disorder.
[0093] In some embodiments, a chronic complement-mediated disorder
affects the circulatory system. For example, in some embodiments
the disorder is a vasculitis or other disorder associated with
vessel inflammation, e.g., blood vessel and/or lymph vessel
inflammation. In some embodiments, a vasculitis is polyarteritis
nodosa, Wegener's granulomatosis, giant cell arteritis,
Churg-Strauss syndrome, microscopic polyangiitis, Henoch-Schonlein
purpura, Takayasu's arteritis, Kawasaki disease, or Behcet's
disease. In some embodiments, a subject, e.g., a subject in need of
treatment for vasculitis, is positive for antineutrophil
cytoplasmic antibody (ANCA).
[0094] In some embodiments, a chronic complement-mediated disorder
affects the gastrointestinal system. For example, the disorder may
be inflammatory bowel disease, e.g., Crohn's disease or ulcerative
colitis. In some embodiments, the invention provides a method of
treating a chronic complement-mediated disorder that affects the
gastrointestinal system, the method comprising administering a
complement inhibitor according to a dosing schedule described
herein to a subject in need of treatment for the disorder.
[0095] In some embodiments, a chronic complement-mediated disorder
is a thyroiditis (e.g., Hashimoto's thryoiditis, Graves' disease,
post-partum thryoiditis), myocarditis, hepatitis (e.g., hepatitis
C), pancreatitis, glomerulonephritis (e.g., membranoproliferative
glomerulonephritis or membranous glomerulonephritis), or
panniculitis.
[0096] In some embodiments, the invention provides methods of
treating a subject suffering from chronic pain, the methods
comprising administering a complement inhibitor to a subject
according to a dosing schedule of the present invention. In some
embodiments, a subject suffers from neuropathic pain. Neuropathic
pain has been defined as pain initiated or caused by a primary
lesion or dysfunction in the nervous system, in particular, pain
arising as a direct consequence of a lesion or disease affecting
the somatosensory system. For example, neuropathic pain may arise
from lesions that involve the somatosensory pathways with damage to
small fibres in peripheral nerves and/or to the
spino-thalamocortical system in the CNS. In some embodiments,
neuropathic pain arises from autoimmune disease (e.g., multiple
sclerosis), metabolic disease (e.g., diabetes), infection (e.g.,
viral disease such as shingles or HIV), vascular disease (e.g.,
stroke), trauma (e.g., injury, surgery), or cancer. For example,
neuropathic pain can be pain that persists after healing of an
injury or after cessation of a stimulus of peripheral nerve endings
or pain that arises due to damage to nerves. Exemplary conditions
of or associated with neuropathic pain include painful diabetic
neuropathy, post-herpetic neuralgia (e.g., pain persisting or
recurring at the site of acute herpes zoster 3 or more months after
the acute episode), trigeminal neuralgia, cancer related
neuropathic pain, chemotherapy-associated neuropathic pain,
HIV-related neuropathic pain (e.g., from HIV neuropathy),
central/post-stroke neuropathic pain, neuropathy associated with
back pain, e.g., low back pain (e.g., from radiculopathy such as
spinal root compression, e.g., lumbar root compression, which
compression may arise due to disc herniation), spinal stenosis,
peripheral nerve injury pain, phantom limb pain, polyneuropathy,
spinal cord injury related pain, myelopathy, and multiple
sclerosis. In certain embodiments of the invention a complement
inhibitor is administered according to an inventive dosing schedule
to treat neuropathic pain in a subject with one or more of the
afore-mentioned conditions.
[0097] In some embodiments, a chronic complement-mediated disorder
is a chronic eye disorder. In some embodiments, the chronic eye
disorder is characterized by macular degeneration, choroidal
neovascularization (CNV), retinal neovascularization (RNV), ocular
inflammation, or any combination of the foregoing. Macular
degeneration, CNV, RNV, and/or ocular inflammation may be a
defining and/or diagnostic feature of the disorder. Exemplary
disorders that are characterized by one or more of these features
include, but are not limited to, macular degeneration related
conditions, diabetic retinopathy, retinopathy of prematurity,
proliferative vitreoretinopathy, uveitis, keratitis,
conjunctivitis, and scleritis. Macular degeneration related
conditions include, e.g., age-related macular degeneration (AMD).
In some embodiments, a subject is in need of treatment for wet AMD.
In some embodiments, a subject is in need of treatment for dry AMD.
In some embodiments, a subject is in need of treatment for
geographic atrophy (GA). In some embodiments, a subject is in need
of treatment for ocular inflammation. Ocular inflammation can
affect a large number of eye structures such as the conjunctiva
(conjunctivitis), cornea (keratitis), episclera, sclera
(scleritis), uveal tract, retina, vasculature, and/or optic nerve.
Evidence of ocular inflammation can include the presence of
inflammation-associated cells such as white blood cells (e.g.,
neutrophils, macrophages) in the eye, the presence of endogenous
inflammatory mediator(s), one or more symptoms such as eye pain,
redness, light sensitivity, blurred vision and floaters, etc.
Uveitis is a general term that refers to inflammation in the uvea
of the eye, e.g., in any of the structures of the uvea, including
the iris, ciliary body or choroid. Specific types of uveitis
include iritis, iridocyclitis, cyclitis, pars planitis and
choroiditis. In some embodiments, a subject is in need of treatment
for geographic atrophy (GA). In some embodiments, the chronic eye
disorder is an eye disorder characterized by optic nerve damage
(e.g., optic nerve degeneration), such as glaucoma.
[0098] In some embodiments, a chronic complement-mediated disorder
is chronic rejection of a transplanted organ, tissue, cells or
populations of cells (collectively "grafts"). Examples of grafts
include, e.g., solid organs such as kidney, liver, lung, pancreas,
heart; tissues such as cartilage, tendons, cornea, skin, heart
valves, and blood vessels; pancreatic islets or islet cells.
Transplant rejection is one of the major risks associated with
transplants between genetically different individuals of the same
species (allografts) or between individuals of different species
(xenografts) and can lead to graft failure and a need to remove the
graft from the recipient. As used herein, "chronic rejection"
refers to rejection occurring at least 6 months post-transplant,
e.g., between 6 months and 1, 2, 3, 4, 5 years, or more
post-transplant, often after months to years of good graft
function. For purposes hereof, chronic rejection can include
chronic graft vasculopathy, a term used to refer to fibrosis of the
internal blood vessels of the transplanted tissue. In some
embodiments, the invention provides a method of treating a subject
in need of treatment to inhibit chronic rejection of a graft, the
method comprising administering a complement inhibitor to the
subject according to a dosing schedule described herein. In some
embodiments, the invention provides a method of treating a subject
who has undergone a transplant or is scheduled to undergo a
transplant within the subsequent 12 weeks. In some embodiments,
treatment is initiated no later than 1, 2, 3, 6, or 12 months
following the transplant.
[0099] In some aspects, the invention provides a method of treating
a subject in need of treatment for a chronic complement-mediated
disorder, e.g., a chronic respiratory disorder, the method
comprising administering multiple doses of a complement inhibitor
to the subject according to a dosing schedule in which successive
doses are administered on average (i) at least 2 weeks after the
plasma concentration of the complement inhibitor decreases to no
more than 20% of the maximum plasma concentration that was reached
after the previous dose; (ii) at least 2 weeks after plasma
complement activation capacity has returned to at least 50% of
baseline or to within the normal range after the previous dose;
(iii) at intervals equal to at least 2 times the terminal plasma
half-life of the complement inhibitor; or (iv) at intervals at
least 3 weeks apart. In some embodiments, an inventive method
comprises administration of a complement inhibitor with an average
dosing interval of at least 3 weeks, e.g., between 3 and 15 weeks,
e.g., between 3 and 12 weeks, e.g., between 3 and 10 weeks, e.g,
between 4 and 8 weeks, e.g., about every 4, 5, 6, 7, or 8 weeks. In
some embodiments, at least 2 of the foregoing conditions are met.
In some embodiments, at least 3 of the foregoing conditions are
met. In some embodiments, all of the foregoing conditions are
met.
[0100] In certain embodiments of the invention, a complement
inhibitor is administered according to a dosing schedule that is
selected based at least in part on local complement activation
capacity and/or local concentration of the complement inhibitor.
For purposes of the present invention, "local complement activation
capacity" refers to complement activation capacity in a tissue or
organ affected by a complement-mediated disorder, which may be
determined, for example, using a relevant sample obtained from such
tissue or organ. For purposes of the present invention, "local
concentration", e.g., local concentration of a complement inhibitor
or a Th17 biomarker such as a Th17-associated cytokine, refers to
concentration in a tissue or organ (e.g., a tissue or organ
affected by a complement-mediated disorder) which may be
determined, for example, using a relevant sample obtained from such
tissue or organ. In some embodiments, a sample comprises a body
fluid obtained from a tissue or organ (or portion thereof) affected
by a complement-mediated disorder. In some embodiments, a fluid is
BAL fluid, sputum (e.g., induced sputum), pleural fluid, synovial
fluid, vitreous or aqueous humor, or cerebrospinal fluid. The
invention provides variations of any of the methods described
herein, in which local complement activation capacity is used
instead of, or in addition to, plasma complement activation
capacity. For example, in certain embodiments of the invention, a
complement inhibitor is administered at least 2 weeks after local
complement activation capacity has returned to at least 50% of
baseline or to within the normal range following the previous dose.
In some embodiments of the invention, a complement inhibitor is
administered between 2 and 15 weeks after local complement
activation capacity has returned to at least 50% of baseline or to
within the normal range following the previous dose. In some
embodiments, a complement inhibitor is administered according to a
dosing schedule in which successive doses are administered on
average (i) at least 2 weeks after the local concentration of the
complement inhibitor decreases to no more than 20% of the maximum
local concentration that was reached after the previous dose In
some embodiments of any of the afore-mentioned methods, a
complement inhibitor is administered locally.
[0101] In some embodiments, an inventive method comprises
administration of a complement inhibitor with an average dosing
interval of at least 3 weeks, e.g., between 3 and 15 weeks, e.g.,
between 3 and 12 weeks e.g., between 3 and 10 weeks, e.g, between 4
and 8 weeks, e.g., about every 4, 5, 6, 7, or 8 weeks. In some
embodiments, an inventive method comprises administration of a
complement inhibitor with an average dosing interval of between 4
and 6 weeks. In some embodiments, a dose sufficient to
substantially inhibit plasma complement activation capacity is
administered. In some embodiments, a dose sufficient to
substantially inhibit local complement activation capacity in a
tissue or organ affected by a complement-mediated disorder is
administered. In some embodiments, complement activation capacity,
e.g., plasma complement activation capacity or local complement
activation capacity, is considered "substantially inhibited" if
reduced to no more than twice background levels, e.g., to
approximately background levels. Background levels (e.g., for any
aspect or embodiment of the invention) may be levels determined
using a variety of suitable approaches. For example, a control
sample, e.g., a control plasma sample or other body fluid sample,
in which complement has been inactivated, e.g., by heat
inactivation, or that has been depleted of one or more complement
components such as C3 can be used, and/or a control assay can be
performed in which an essential assay component is omitted. In some
embodiments, a dose sufficient to reduce and/or maintain plasma
complement to within the normal range administered. In some
embodiments, a dose sufficient to reduce and/or maintain local
complement activation in a tissue or organ affected by a
complement-mediated disorder to within the normal range is
administered.
[0102] In some embodiments of an inventive method, element (i)
comprises administering multiple doses of a complement inhibitor to
the subject according to a dosing schedule in which successive
doses are administered on average at least 2 weeks after the plasma
concentration of the complement inhibitor decreases to no more than
10%, or in some embodiments no more than 5%, or in some embodiments
no more than 1%, of the maximum plasma concentration that was
reached after the previous dose. In some embodiments of an
inventive method, element (i) comprises administering multiple
doses of a complement inhibitor to the subject according to a
dosing schedule in which successive doses are administered on
average at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
weeks after the plasma concentration of the complement inhibitor
decreases to no more than 20% of the maximum plasma concentration
that was reached after the previous dose or, in some embodiments at
least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks after
the plasma concentration of the complement inhibitor decreases to
no more than 10%, or in some embodiments no more than 5%, or in
some embodiments no more than 1%, of the maximum plasma
concentration that was reached after the previous dose.
[0103] In some embodiments, an inventive method comprises
administering a complement inhibitor at intervals such that the
subject's plasma complement activation capacity is at least 50% of
baseline or within the normal range for on average at least 2 weeks
between doses. In some embodiments, an inventive method comprises
administering a complement inhibitor at intervals such that the
subject's plasma complement activation capacity is at least 50% of
baseline for on average at least 2 weeks between doses. "Baseline"
in this context refers to the subject's typical complement
activation capacity when not affected by administration of an agent
or exposure to a stimulus that significantly affects the complement
system; and not having experienced an exacerbation of asthma or
COPD (or, in some aspects of the invention, another
complement-mediated disorder, as applicable) within the preceding 6
weeks. In some embodiments, an inventive dosing regimen comprises
administering a complement inhibitor at intervals such that the
subject's plasma complement activation capacity is within the
normal range for on average at least 2 weeks between doses. "Normal
range" in this context typically refers to a range of within .+-.2
standard deviations from a mean value (e.g., an arithmetic mean
value) in a population of subjects. One of ordinary skill in the
art will appreciate that the specific values for a "normal range"
may at least in part depend on the particular assay used to assess
complement activation capacity and/or factors such as the specific
reagents used. In some embodiments, a normal range may be
determined using published data. In some embodiments, a normal
range may be appropriately defined by a laboratory, testing center,
ordinary skilled artisan, etc.
[0104] In some embodiments, the complement inhibitor is
administered with a dosing interval such that the subject's
complement activation capacity is at least 50% of baseline or
within the normal range for on average at least 3 weeks, e.g.,
between 3 and 15 weeks, e.g., e.g., between 3 and 12 weeks, e.g.,
between 3 and 10 weeks, e.g, between 4 and 8 weeks, e.g., about 4,
5, 6, 7, or 8 weeks between doses. For purposes of the present
invention, it will be assumed that plasma and serum complement
activation capacity are not significantly different and can be used
interchangeably absent evidence to the contrary. If a difference is
determined to exist, the invention provides embodiments in which
plasma complement activation capacity is used, embodiments in which
the serum complement activation capacity is used, and embodiments
in which an average value is used.
[0105] In some embodiments, an inventive method comprises
administering a complement at intervals at least equal on average
to twice (2.times.) the plasma half-life of the complement
inhibitor when administered intravenously. In some embodiments, an
inventive dosing regimen comprises administering a complement
inhibitor at intervals at least equal on average to 3.times.,
4.times., 5.times., 6.times., 7.times., 8.times., 9.times., or
10.times. the plasma half-life of the complement inhibitor when
administered intravenously. In some embodiments an inventive method
comprises administering a complement inhibitor by a selected
administration route, at intervals at least equal on average to
twice (2.times.) the plasma half-life of the complement inhibitor
when administered by the same route. In some embodiments, an
inventive method comprises administering a complement inhibitor at
intervals at least equal on average to 3.times., 4.times.,
5.times., 6.times., 7.times., 8.times., 9.times., or 10.times. the
plasma half-life of the complement inhibitor when administered by
the same route. In some embodiments, an administration route is the
respiratory route. In some embodiments, a complement inhibitor has
a mean plasma half-life of between 1 and 5 days. In some
embodiments, a complement inhibitor has a mean plasma half-life of
between 5 and 10 days. In some embodiments, a complement inhibitor
has a mean plasma half-life of between 10 and 20 days. In some
embodiments, a complement inhibitor has a mean plasma half-life of
between 20 and 30 days.
[0106] It will be appreciated that a variety of approaches to
determining pharmacokinetic (PK) parameters such as half-life can
be used. An appropriate method can be selected by one of ordinary
skill in the art. In general, half-life can be determined by a
method comprising: administering one or more doses of the compound
to subjects, obtaining blood samples from the subject at various
times after administration, measuring the concentration of the
compound in said samples, and calculating a half-life based at
least in part on said measurements. For example, in some
embodiments, samples may be obtained at times 0 (pre-dose), 5 min,
15 min, 30 min, 1 hr, 4 hr, 8 hr, 24 hr (1 day), 48 hr (2 days), 96
hr (4 days), 192 hr (8 days), 14 days, 21 days, 28 days post-dose.
It will be appreciated that these time points are exemplary.
Different time points and/or more or fewer time points could be
used in various embodiments. One of ordinary skill in the art would
select appropriate time points. The blood samples are typically
processed to obtain plasma or serum prior to making the
measurements. For purposes of the present invention, it will be
assumed that plasma and serum concentrations (and pharmacokinetic
parameters such as half-life) are not significantly different and
can be used interchangeably absent evidence to the contrary. If a
difference is determined to exist, the invention provides
embodiments in which plasma concentrations (and/or plasma
half-life) is used, embodiments in which the serum concentrations
(and/or serum half-life therein) is used, and embodiments in which
an average value is used.
[0107] One of ordinary skill in the art would select an appropriate
method for measuring the compound. For example, in some embodiments
an immunoassay is used. In some embodiments, a chromatography-based
method is used (e.g., liquid chromatography (LC), liquid
chromatography-mass spectrometry (LC-MS) or liquid
chromatography-tandem mass spectrometry (LC-MS-MS). In some
embodiments, a bioassay is used. In many embodiments, the half-life
is a terminal (elimination) half-life. In some embodiments, a
terminal half-life is calculated following administration of a
single dose. In some embodiments, a terminal half-life is
calculated following administration of multiple doses and allowing
the concentration to reach steady state. In some embodiments, a
half-life determined for the initial (distribution) phase is used.
For example, if the majority of the compound is removed from
circulation during the distribution phase, an initial half-life may
be used in some embodiments.
[0108] In some embodiments, half-life is determined by conducting a
PK analysis using non-compartmental analysis on multiple dose PK
data from a group of subjects. In some embodiments, half-life is
determined by conducting a PK analysis using a standard
1-compartment model on multiple dose PK data from a group of
subjects. In some embodiments, a half-life determined in subjects
suffering from a chronic respiratory disorder (e.g., asthma or
COPD) is used. In some embodiments, a half-life determined in
subjects who are healthy and not known to be suffering from a
disorder is used. In some embodiments, a half-life determined in
subjects suffering from a complement-mediated disorder other than a
chronic respiratory disorder is used. In some embodiments, a
half-life determined in adults (persons at least 18 years of age)
is used.
[0109] In some embodiments, half-life is determined using a dose
suitable for treating a chronic complement-mediated disorder, e.g.,
a chronic respiratory disorder, e.g., asthma or COPD. In some
embodiments, a dose is sufficient to reduce plasma complement
activation capacity to no more than 50% of the lower limit of the
normal range. In some embodiments, a dose is sufficient to reduce
plasma complement activation capacity to no more than twice
background levels, e.g., to approximately background levels. In
some embodiments, half-life is determined using a composition
comprising the complement inhibitor, wherein the composition is the
same or substantially similar to a composition to be used to treat
a chronic complement-mediated disorder.
[0110] In certain embodiments, a complement inhibitor is modified
by conjugation with a polypeptide or non-polypeptide component of
use to stabilize the compound, reduce its immunogenicity, increase
its lifetime in the body, increase or decrease its solubility,
and/or increase its resistance to degradation. For example, a
polymer such as polyethylene glycol (PEG), albumin, or
albumin-binding peptide, may be used. In such embodiments,
"half-life" typically refers to the half-life of the complement
inhibitor as so modified.
[0111] A variety of software tools are available to facilitate
calculation of PK parameters. For example, Phoenix NMLE or Phoenix
WinNonlin software (PharSight Corp, St. Louis, Mo.) or Kinetica
(Thermo Scientific) can be used. It will be appreciated that a
reasonable estimate of half-life based on a model can be used. In
some embodiments, a half-life determined in a Phase I, II, or III
clinical trial of a particular compound and/or submitted in an
application to a regulatory agency such as the FDA (e.g., an IND or
NDA) is used as a half-life in determining an inventive dosing
interval.
[0112] In some embodiments, a method comprises administering at
least 5, 10, 15, 20, or 25 doses are to a subject according to an
inventive dosing schedule (i.e., using a dosing interval according
to the invention). In some embodiments, treatment is continued over
a period at least 3, 6, 9, 12 months, or more, e.g., 1-2 years, 2-5
years, 5-10 years, or more, e.g., indefinitely.
[0113] It will be appreciated that minor deviations, such as
occasional use of a shorter or longer dosing interval as compared
with a dosing interval or range specified herein (e.g., up to about
5%, 10%, or 20% of doses, e.g., within a time span such as 6
months, 1 year, etc.) would fall within the scope of the invention.
In some embodiments, a dosing interval for a subject may vary over
time and/or may be selected at least in part based on a measurement
of complement activation capacity and/or assessment of disease
activity (or a biomarkerthereof) between doses.
[0114] In some embodiments of any of the inventive methods, a
complement inhibitor is administered intravenously. In some
embodiments of any of the inventive methods, a complement inhibitor
is administered by the respiratory route. In some embodiments of
any of the inventive methods, a complement inhibitor is
administered subcutaneously. In some embodiments of any of the
inventive methods, a complement inhibitor is administered
intramuscularly. In some embodiments of any of the inventive
methods, a complement inhibitor is administered orally.
[0115] In some embodiments, a complement inhibitor is administered
in a formulation that provides sustained release (also referred to
as "extended release" or "controlled release") of the complement
inhibitor. In some embodiments in which a sustained release
formulation is used, the time interval between doses is calculated
based at least in part on the length of time that the sustained
release formulation releases complement inhibitor. For example, if
a sustained release formulation releases a complement inhibitor for
N weeks after administration before becoming depleted, the
invention provides a method of treating a subject comprising
administering multiple doses of said sustained release formulation
according to a dosing schedule in which successive doses are
administered with an average dosing interval of at least N+3 weeks,
e.g., between N+3 and N+15 weeks, e.g., between N+3 and N+12 weeks,
e.g., between N+3 and N+10 weeks, e.g, between N+4 and N+8 weeks,
e.g., about every N+4, N+5, N+6, N+7, or N+8 weeks. In some
embodiments, a sustained release formulation is considered to be
depleted if it no longer releases sufficient complement inhibitor
to maintain the subject's plasma complement activation capacity
and/or local complement activation capacity (e.g., in a tissue or
organ affected by a complement-mediated disorder) below the normal
range or reduced by at least 50% of baseline. In some embodiments,
a sustained release formulation is considered to be depleted if it
no longer releases sufficient complement inhibitor to maintain the
subject's plasma complement activation and/or local complement
activation (e.g., in a tissue or organ affected by a
complement-mediated disorder) below the normal range or reduced by
at least 50% of baseline. In some embodiments, a sustained release
formulation is considered to be depleted if at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or more of the complement inhibitor
contained in the formulation when administered has been released or
the formulation has essentially ceased releasing complement
inhibitor.
[0116] All combinations of the various complement inhibitors,
complement inhibitor characteristics (e.g., compound class,
molecular weight, half-life, molecular target, etc.), and dosing
parameters (e.g., dosing interval, route of administration, etc.),
and disorders, e.g., respiratory disorders disclosed herein are
contemplated in various embodiments of the invention. For example,
in some embodiments, an inventive method comprises intravenous
administration of a complement inhibitor with an average dosing
interval of at least 3 weeks, e.g., between 3 and 15 weeks, e.g.,
between 3 and 12 weeks, e.g., between 3 and 10 weeks, e.g, between
4 and 8 weeks, e.g., about every 4, 5, 6, 7, or 8 weeks. In some
embodiments, an inventive method comprises pulmonary administration
of a complement inhibitor with an average dosing interval of at
least 3 weeks, e.g., e.g., between 3 and 15 weeks, e.g., between 3
and 12 weeks, e.g., between 3 and 10 weeks, e.g, about every 4, 5,
6, 7, or 8 weeks.
[0117] Further provided are methods of selecting a dosing interval
for administering a complement inhibitor. In some embodiments, a
method of selecting a dosing interval for administering a
complement inhibitor comprises (a) obtaining a half-life of the
complement inhibitor; and (b) selecting a dosing interval at least
2-10 weeks longer than the half-life. In some embodiments, a method
of selecting a dosing interval for administering a complement
inhibitor comprises (a) obtaining a half-life of the complement
inhibitor; and (b) selecting a dosing interval at least 3 times as
long as the half-life. In some embodiments, a method of selecting a
dosing interval for a complement inhibitor comprises: (a)
determining the length of time that the complement inhibitor
reduces plasma complement activation capacity by at least 50% of
baseline and/or the length of time that the complement inhibitor
reducees plasma complement activation capacity to below the normal
range; and (b) selecting any of the inventive dosing intervals set
forth above based on said measured length of time. In some
embodiments, a method of selecting a dosing interval can further
comprise testing a complement inhibitor administered according to
an inventive dosing schedule to an animal that serves as a model
for a chronic complement-mediated disorder, e.g., a chronic
complement-mediated respiratory disorder.
[0118] In some embodiments, an inventive treatment method comprises
an induction phase and a maintenance phase. In many embodiments,
the induction phase (if used) occurs when a subject initiates
therapy. The induction phase can consist of a period of time during
which a complement inhibitor is administered at a higher dose
and/or at more frequent intervals and/or using a different route of
administration than during the maintenance phase. During the
maintenance phase, the complement inhibitor may be administered
using any of the inventive dosing schedules and/or dosing intervals
described above. For example, the complement inhibitor may be
administered weekly during an induction phase and on average every
4-15 weeks, e.g., every 4-8 weeks, during a maintenance phase. In
some embodiments a complement inhibitor is administered once or
more times daily during an induction phase. In some embodiments a
complement inhibitor is administered at least 1, 2, 3, 4, 5, 6, or
7 times weekly during an induction phase. In some embodiments an
induction phase lasts for up to 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In
some embodiments a dose or dosing interval is adjusted during an
induction phase. For example, in some embodiments the dosing
interval may be increased over time and/or the dose may be
decreased or increased over time during the induction phase.
[0119] As noted above, in some embodiments the chronic respiratory
disease is asthma. Information regarding risk factors,
epidemiology, pathogenesis, diagnosis, current management of
asthma, etc., may be found, e.g., in "Expert Panel Report 3:
Guidelines for the Diagnosis and Management of Asthma". National
Heart Lung and Blood Institute. 2007.
http://www.nhlbi.nih.gov/guidelines/asthma/asthgdln.pdf. ("NHLBI
Guidelines"; www.nhlbi.nih.gov/guidelines/asthma/asthgdln.htm),
Global Initiative for Asthma, Global Strategy for Asthma Management
and Prevention 2010 "GINA Report") and/or standard textbooks of
internal medicine such as Cecil Textbook of Medicine (20th
edition), Harrison's Principles of Internal Medicine (17th
edition), and/or standard textbooks focusing on pulmonary medicine.
Asthma is a chronic inflammatory disorder of the airways in which
many cells and cellular elements play a role, such as, mast cells,
eosinophils, T lymphocytes, macrophages, neutrophils, and
epithelial cells Asthmatic individuals experience recurrent
episodes associated with symptoms such as wheezing, breathlessness
(also termed dyspnea or shortness of breath), chest tightness, and
coughing. These episodes are usually associated with widespread but
variable airflow obstruction that is often reversible, either
spontaneously or with treatment. The inflammation also causes an
associated increase in the existing bronchial hyperresponsiveness
to a variety of stimuli. Airway hyperresponsiveness (an exaggerated
bronchoconstrictor response to stimuli) is a typical feature of
asthma. In general, airflow limitation results from
bronchoconstriction and airway edema. Reversibility of airflow
limitation may be incomplete in some patients with asthma. For
example, airway remodeling can lead to fixed airway narrowing.
Structural changes can include thickening of the sub-basement
membrane, subepithelial fibrosis, airway smooth muscle hypertrophy
and hyperplasia, blood vessel proliferation and dilation, and
mucous gland hyperplasia, and hypersecretion.
[0120] Individuals with asthma may experience exacerbations, which
are identified as events characterized by a change from the
individual's previous status. Severe asthma exacerbations can be
defined as events that require urgent action on the part of the
individual and his/her physician to prevent a serious outcome, such
as hospitalization or death from asthma. For example, a severe
asthma exacerbation may require use of systemic corticosteroids
(e.g., oral corticosteroids) in a subject whose asthma is usually
well controlled without OCS or may require an increase in a stable
maintenance dose. Moderate asthma exacerbations can be defined as
events that are troublesome to the subject, and that prompt a need
for a change in treatment, but that are not severe. These events
are clinically identified by being outside the subject's usual
range of day-to-day asthma variation.
[0121] Current medications for asthma are typically categorized
into two general classes: long-term control medications
("controller medications") such as inhaled corticosteroids (ICS),
oral corticosteroids (OCS), long-acting bronchodilators (LABAs),
leukotriene modifiers (e.g., leukotriene receptor antagonists or
leukotriene synthesis inhibitors, anti-IgE antibodies (omalizumab
(Xolair.RTM.)), cromolyn and nedocromil, which are used to achieve
and maintain control of persistent asthma and quick-relief
medications such as short-acting bronchodilators (SABAs), which are
used to treat acute symptoms and exacerbations. For purposes of the
present invention, these treatments may be referred to as
"conventional therapy". Treatment of exacerbations may also include
increasing the dose and/or intensity of controller medication
therapy. For example, a course of OCS can be used to regain asthma
control. Current guidelines mandate daily administration of
controller medication or, in many cases, administration of multiple
doses of controller medication each day for subjects with
persistent asthma (with the exception of Xolair, which is
administered every 2 or 4 weeks).
[0122] A subject is generally considered to have persistent asthma
if the subject suffers from symptoms on average more than twice a
week and/or typically uses a quick relief medication (e.g., SABA)
more than twice a week for symptom control. "Asthma severity" can
be classified based on the intensity of treatment required to
control the subject's asthma once relevant comorbidities have been
treated and inhaler technique and adherence have been optimized
(see, e.g., GINA Report; Taylor, D R, Eur Respir J 2008;
32:545-554). The description of treatment intensity can be based on
the medications and doses recommended in the stepwise treatment
algorithm found in guidelines such as NHLBI Guidelines 2007, GINA
Report, and their predecessors and/or in standard medical
textbooks. For example, asthma can be classified as intermittent,
mild, moderate, or severe as indicated in Table 1, where
"treatment" refers to treatment sufficient to achieve subject's
best level of asthma control. (It will be understood that the
categories of mild, moderate, and severe asthma in general imply
persistent rather than intermittent asthma). One of ordinary skill
in the art will appreciate that Table 1 is exemplary, and that not
all of these medications will be available in all healthcare
systems, which may affect the assessment of asthma severity in some
environments. It will also be appreciated that other emerging or
new approaches may affect the classification of mild/moderate
asthma. However, the same principle, of mild asthma being defined
by the ability to achieve good control using very low-intensity
treatment and severe asthma being defined by the requirement for
high-intensity treatment, can still be applied. Asthma severity can
also or alternately be classified based on intrinsic intensity of
the disease in the absence of treatment (see, e.g., NHBLI
Guidelines 2007). Assessment can be made on the basis of current
spirometry and the patient's recall of symptoms over the previous
2-4 weeks. Parameters of current impairment and future risk may be
assessed and included in a determination of the level of asthma
severity. In some embodiments, asthma severity is defined as shown
in FIG. 3.4(a), 3.4(b), 3.4(c) of the NHBLI Guidelines, for
individuals 0-4, 5-11, or .gtoreq.12 years of age,
respectively.
TABLE-US-00001 TABLE 1 Treatment-based Asthma Classification Asthma
Classification Treatment Intermittent SABA as needed (typically no
more than twice a week) Mild Low-dose ICS or other low-intensity
treatment (e.g., LTRA, cromolyn, nedocromil, theophylline) Moderate
Low to moderate dose ICS and LABA or other extra treatment Severe
High-intensity treatment (high-dose ICS and LABA .+-. oral
corticosteroids and/or other extra treatment)
[0123] "Asthma control" refers to the extent to which the
manifestations of asthma have been reduced or removed by treatment
(whether pharmacological or non-pharmacological). Asthma control
can be assessed based on factors such as symptom frequency,
nighttime symptoms, objective measures of lung function such as
spirometry parameters (e.g., % FEV.sub.1 of predicted, FEV.sub.1
variability, requirement for use of SABA for symptom control.
Parameters of current impairment and future risk may be assessed
and included in a determination of the level of asthma control. In
some embodiments, asthma control is defined as shown in FIG.
4.3(a), 4.3(b), or 4.3(c) of NHBLI Guidelines, for individuals 0-4,
5-11, or .gtoreq.12 years of age, respectively.
[0124] In general, one of ordinary skill in the art can select an
appropriate means of determining asthma severity level and/or
degree of control, and any classification scheme considered
reasonable by those of ordinary skill in the art can be used.
[0125] In some embodiments of the invention, a subject suffering
from persistent asthma is treated with a complement inhibitor using
an inventive dosing regimen. In some embodiments, the subject
suffers from mild or moderate asthma. In some embodiments, the
subject suffers from severe asthma. In some embodiments, a subject
has asthma that is not well controlled using conventional therapy.
In some embodiments, a subject has asthma that, when treated using
conventional therapy, requires use of ICS in order to be well
controlled. In some embodiments, a subject has asthma that fails to
be well controlled despite use of ICS. In some embodiments, a
subject has asthma that, if treated using conventional therapy,
would require use of OCS in order to be well controlled. In some
embodiments, a subject has asthma that fails to be well controlled
despite use of high intensity conventional therapy that includes
OCS. In some embodiments of the invention, an inventive dosing
regimen comprises administering a complement inhibitor as a
controller medication, wherein the complement inhibitor is
administered with reduced frequency and/or on a less regular basis,
as compared with standard controller medications, while maintaining
at least equivalent asthma control. In some embodiments, an
inventive dosing regimen affords improved patient acceptability,
compliance, and/or convenience, as compared with standard regimens
of conventional controller medications, while maintaining at least
equivalent asthma control. In some embodiments, a subject treated
with a complement inhibitor, e.g., according to an inventive dosing
regimen, can significantly decrease the dose (e.g., by at least
50%) or substantially avoid use of ICS, Xolair, and/or OCS as a
controller medication.
[0126] In some embodiments, the subject suffers from allergic
asthma, which is the case for most asthmatic individuals. In some
embodiments, an asthmatic subject is considered to have allergic
asthma if a non-allergic trigger for the asthma (e.g., cold,
exercise) is not known and/or is not identified in a standard
diagnostic evaluation. In some embodiments, an asthmatic subject is
considered to have allergic asthma if the subject (i) reproducibly
develops asthma symptoms (or worsening of asthma symptoms)
following exposure to an allergen or allergen(s) to which the
subject is sensitive; (ii) exhibits IgE specific for an allergen or
allergen(s) to which the subject is sensitive; (iii) exhibits a
positive skin-prick test to an allergen or allergen(s) to which the
subject is sensitive; and/or (iv) exhibits other symptom(s) of
characteristic(s) consistent with atopy such as allergic rhinitis,
eczema, or elevated total serum IgE. It will be appreciated that a
specific allergic trigger may not be identified but may be
suspected or inferred if the subject experiences worsening symptoms
in particular environments, for example.
[0127] Allergen challenge by inhalation is a technique that is
widely used in evaluating allergic airway disease. Inhalation of
allergen leads to cross-linking of allergen-specific IgE bound to
IgE receptors on, e.g., mast cells and basophils. Activation of
secretory pathways ensues, resulting in release of mediators of
bronchoconstriction and vascular permeability. Individuals with
allergic asthma may develop various manifestations following
allergen challenge, e.g., early asthmatic response (EAR), late
asthmatic response (LAR), airway hyperreactivity (AHR), and airway
eosinophilia, each of which can be detected and quantified as known
in the art. For example, airway eosiphophilia may be detected as an
increase in eosinophils in sputum and/or BAL fluid. The EAR,
sometimes referred to as the immediate asthmatic response (IAR), is
a response to allergen challenge by inhalation that becomes
detectable shortly after the inhalation, typically within 10
minutes (min) of the inhalation, e.g., as a decrease in FEV.sub.1.
The EAR typically reaches a maximum within 30 min and resolves
within 2-3 hours (h) post-challenge. For example, a subject may be
considered to exhibit a "positive" EAR if his/her FEV.sub.1
decreases by at least 15%, e.g., at least 20%, within this time
window relative to baseline FEV.sub.1 (where "baseline" in this
context refers to conditions before the challenge, e.g., conditions
equivalent to the subject's usual condition when not experiencing
an asthma exacerbation and not exposed to allergic stimuli to which
the subject is sensitive). The late asthmatic response (LAR)
typically starts between 3 h and 8 h post-challenge and is
characterized by cellular inflammation of the airway, increased
bronchiovascular permeability, and mucus secretion. It is typically
detected as a decrease in FEV.sub.1, which may be greater in
magnitude than that associated with the EAR and potentially more
clinically important. For example, a subject may be considered to
exhibit a "positive" LAR if his/her FEV.sub.1 decreases by at least
15%, e.g., at least 20%, relative to baseline FEV.sub.1 within the
relevant time period as compared with baseline FEV.sub.1. A delayed
airway response (DAR) may occur beginning between about 26 and 32
h, reaching a maximum between about 32 and 48 h and resolving
within about 56 h after the challenge (Pelikan, Z. Ann Allergy
Asthma Immunol. 2010, 104(5):394-404).
[0128] In some embodiments, the chronic respiratory disorder is
chronic obstructive pulmonary disease (COPD). COPD encompasses a
spectrum of conditions characterized by airflow limitation that is
not fully reversible even with therapy and is usually progressive.
Symptoms of COPD include dyspnea (breathlessness), decreased
exercise tolerance, cough, sputum production, wheezing, and chest
tightness. Persons with COPD can experience episodes of acute
(e.g., developing over course of less than a week and often over
the course of 24 hours or less) worsening of symptoms (termed COPD
exacerbations) that can vary in frequency and duration and are
associated with significant morbidity. They may be triggered by
events such as respiratory infection, exposure to noxious
particles, or may have an unknown etiology. Smoking is the most
commonly encountered risk factor for COPD, and other inhalational
exposures can also contribute to development and progression of the
disease. The role of genetic factors in COPD is an area of active
research. A small percentage of COPD patients have a hereditary
deficiency of alpha-1 antitrypsin, a major circulating inhibitor of
serine proteases, and this deficiency can lead to a rapidly
progressive form of the disease.
[0129] Characteristic pathophysiologic features of COPD include
narrowing of and structural changes in the small airways and
destruction of lung parenchyma (in particular around alveoli), most
commonly due to chronic inflammation. The chronic airflow
limitation observed in COPD typically involves a mixture of these
factors, and their relative importance in contributing to airflow
limitation and symptoms varies from person to person. The term
"emphysema" refers to enlargement of the air spaces (alveoli)
distal to the terminal bronchioles, with destruction of their
walls. It should be noted that the term "emphysema" is often used
clinically to refer to the medical condition associated with such
pathological changes. Some individuals with COPD have chronic
bronchitis, which is defined in clinical terms as a cough with
sputum production on most days for 3 months of a year, for 2
consecutive years. Further information regarding risk factors,
epidemiology, pathogenesis, diagnosis, and current management of
COPD may be found, e.g., in "Global Strategy for the Diagnosis,
Management, and Prevention of Chronic Obstructive Pulmonary
Disease" (updated 2009) available on the Global Initiative on
Chronic Obstructive Pulmonary Disease, Inc. (GOLD) website
(www.goldcopd.org), also referred to herein as the "GOLD Report",
the American Thoracic Society/European Respiratory Society
Guidelines (2004) available on the ATS website at
www.thoracic.org/clinical/copd-guidelines/resources/copddoc.pdf,
referred to herein as "ATC/ERS COPD Guidelines" and standard
textbooks of internal medicine such as Cecil Textbook of Medicine
(20.sup.th edition), Harrison's Principles of Internal Medicine
(17.sup.th edition), and/or standard textbooks focusing on
pulmonary medicine.
[0130] In some embodiments methods disclosed herein inhibit
(interfere with, disrupt) the DC-Th17-B-Ab-C-DC cycle discussed
above. For example, administration of a complement inhibitor may
break the cycle by which complement stimulates DC cells to promote
the Th17 phenotype. As a result, the number and/or activity of Th17
cells diminishes, which in turn reduces the amount of Th17-mediated
stimulation of B cells and polyclonal antibody production. In some
embodiments, these effects result in "resetting" the immunological
microenvironment to a more normal, less pathological state. As
described in Example 1, evidence supporting the capacity of
complement inhibition to have a prolonged inhibitory effect on
Th17-associated cytokine production was obtained in an animal model
of asthma.
[0131] In some embodiments, inhibiting the DC-Th17-B-Ab-C-DC cycle
has a disease-modifying effect. Without wishing to be bound by any
theory, rather than merely treating symptoms of a disorder,
inhibiting the DC-Th17-B-Ab-C-DC cycle may interfere with
fundamental pathologic mechanisms that may contribute to ongoing
tissue damage even when symptoms are well controlled and/or that
may contribute to exacerbations of the disease. In some
embodiments, inhibiting the DC-Th17-B-Ab-C-DC cycle causes a
chronic disorder to go into remission. In some embodiments,
remission refers to a state of absence or substantial absence of
disease activity in a subject with a chronic disorder, with the
possibility of return of disease. In some embodiments remission may
be sustained for a prolonged period of time (e.g., at least 6
months, e.g., 6-12 months, 12-24 months, or more) in the absence of
continued therapy or with a reduced dose or increased dosing
interval. In some aspects, inhibition of complement may change the
immunological micro-environment of a tissue that is rich in Th17
cells and modify it into a micro-environment that is rich in
regulatory T cells (Tregs). Doing so could allow the immune system
to "reset" itself and go into a state of remission. In some
embodiments, for example, remission may be sustained until
occurrence of a triggering event. A triggering event may be, for
example, an infection (which may result in production of polyclonal
antibodies that react both with an infectious agent and a self
protein), exposure to particular environmental conditions (e.g.,
high levels of air pollutants such as ozone or particulate matter
or components of smoke such as cigarette smoke, allergens), etc.
Genetic factors may play a role. For example, individuals having
particular alleles of genes encoding complement components may have
a higher baseline level of complement activity, a more reactive
complement system and/or a lower baseline level of endogenous
complement regulatory protein activity. In some embodiments an
individual has a genotype associated with increased risk of AMD.
For example, the subject may have a polymorphism in a gene encoding
a complement protein or complement regulatory protein, e.g., CFH,
C3, factor B, wherein the polymorphism is associated with an
increased risk of AMD.
[0132] In some embodiments an immunologic microenvironment may
become progressively more polarized towards a pathological state
over time, e.g., in a subject who has not yet developed symptoms of
a chronic disorder or in a subject who has developed the disorder
and has been treated as described herein. Such a transition may
occur stochastically (e.g., due at least in part to apparently
random fluctuations in antibody levels and/or affinity) and/or as a
result of accumulated "sub-threshold" trigger events that are not
of sufficient intensity to trigger a symptomatic outbreak of a
disorder.
[0133] In some aspects, methods disclosed herein comprise
monitoring a subject for evidence of the DC-Th17-B-Ab-C-DC cycle.
If such evidence is detected, the subject may be treated with a
complement inhibitor and/or other agent that disrupts the
DC-Th17-B-Ab-C-DC cycle. In some embodiments a subject is tested
for Th17 cells (e.g., Th17 cell number or relative number) and/or
for one or more biomarkers associated with Th17 cells and/or Th17
activity ("Th17 biomarker"). In some embodiments, a subject is
treated with a complement inhibitor based at least in part on
assessment of Th17 cells with a Th17 biomarker. "Th17 biomarker"
encompasses any molecule or detectable indicator that correlates
with Th17 cell presence (e.g., number or concentration of Th17
cells) and/or correlates with at least one Th17 cell activity. In
some embodiments, a Th17 biomarker comprises a level of a
Th17-associated cytokine. In some embodiments a Th17-associated
cytokine is a cytokine that promotes formation and/or activation of
Th17 cells, e.g., IL-6, IL-21, IL-23, and/or IL-113. In some
embodiments a Th17-associated cytokine is a cytokine produced by
Th17 cells, e.g., IL-17 (e.g., IL-17A and/or IL-17F), IL-21, and/or
IL-22. In some embodiments an increased amount or increased
relative amount of a Th17-associated activity is indicative of
increased Th17 cells and/or increased Th17-associated activity. In
some embodiments a relative amount is an amount as compared with a
different cytokine. In some embodiments the different cytokine is
associated with Treg cells. In some embodiments the different
cytokine is IL-10. In some embodiments levels of 2, 3, 4, 5, or
more Th17-associated cytokines are measured. A collective index or
score indicative of the level of Th17-associated activity may be
obtained and used as a Th17 biomarker. In some embodiments the
presence or level of Th17 cells themselves is assessed for any
purpose for which a Th17 biomarker may be assessed. In some
embodiments the presence or level of Tregs is assessed. In some
embodiments Tregs are identified based on expression of FOXP3.
[0134] In some embodiments, a Th17 biomarker level is measured in a
sample obtained from a subject. In some embodiments a sample
comprises a body fluid, e.g., blood, BAL fluid, sputum, nasal
secretion, urine, etc. In some embodiments a sample comprises a
tissue sample, which may be obtained from a tissue or organ
affected by a complement-mediated disorder. In some embodiments two
or more samples of different body fluids or a body fluid and a
tissue sample are assessed. In some embodiments a level is compared
with a reference value. In some embodiments a reference value may
be a normal value (e.g., a value within a normal range, e.g., an
upper limit of a normal range). In some embodiments a reference
value may be a value established for the subject at a previous
time, e.g., when the subject's disorder was well controlled or
prior to development of the disorder. In some embodiments, if a
measured value deviates significantly from a reference value or
shows a trend towards increased deviation from a reference value,
the subject may be treated with a complement inhibitor. In some
embodiments the subject may be treated with a complement inhibitor
and a second agent that disrupts the DC-Th17-B-Ab-C-DC cycle. A
"normal range" may be a range that encompasses at least 95% of
healthy individuals. In some embodiments a reference value may be a
value associated with a disease, e.g., a value typically found in
subjects suffering from a disease in an untreated state. In some
embodiments a normal or disease-associated range may depend at
least in part on demographic factors such as age, sex, etc., and
can be adjusted accordingly. An appropriate reference value or
range may be established empirically for different disorders and/or
different Th17 biomarkers and/or, in some embodiments, for
individual subjects.
[0135] In some embodiments, in vivo assessment of Th17 cells and/or
a Th17 biomarker is envisioned. For example, in some embodiments a
detectably labeled agent that binds to Th17 cells (e.g., to a cell
surface marker or combination thereof that is reasonably specific
for Th17 cells) or that bind to a Th17-associated cytokine is
administered to a subject. A suitable imaging method is used to
visualize the agent in vivo. In some embodiments, for example, an
image is obtained of the lungs, skin, or other location that may be
affected by a complement-mediated disorder. In some embodiments in
vivo detection allows assessment of the immunological
microenvironment in a tissue or organ of interest. In some
embodiments a detectable label comprises a fluorescent,
radioactive, ultrasound, or magnetically detectable moiety. In some
embodiments an imaging method comprises magnetic resonance imaging,
ultrasound imaging, optical imaging (e.g., fluorescence imaging or
bioluminescence imaging), or nuclear imaging. In some embodiments a
fluorescent moiety comprises a near-infrared or infrared
fluorescent moiety (emitting in the near-infrared or infrared
region of the spectrum). In some embodiments an imaging method
comprises positron emission tomography (PET), and single photon
emission computed tomography (SPECT) In some embodiments a
detectable label is attached to an agent that binds directly to a
target to be detected. In some embodiments a detectable label is
associated with or incorporated into or comprises particles, which
in some embodiments have at their surface an agent that binds
directly to a target to be detected.
[0136] In some embodiments, information obtained from a Th17
biomarker assessment is used together with additional information,
e.g., genotype information, environmental exposure information,
and/or subject historical information, to determine whether or when
to administer a complement-inhibitor and/or anti-Th17 agent and/or
to select a dose or dosing regimen for a subject. In some
embodiments any of the biomarker assessment and/or treatment
decision methods may be performed at least in part by one or more
computers. In some embodiments any of the biomarker assessment
and/or treatment decision methods may be embodied or stored at
least in part on a computer-readable medium having
computer-executable instructions thereon. In some embodiments a
computer-readable medium comprises any non-transitory and/or
tangible computer-readable medium.
[0137] In some embodiments retreatment may occur on a fixed time
schedule.
[0138] Wherever an aspect or embodiment herein is described in
relation to complement-mediated disorders, analogous aspects and
embodiments relating to Th17-associated disorders are provided.
Wherever an aspect or embodiment herein is described in relation to
complement-mediated disorders, analogous aspects and embodiments
relating to Th17-associated disorders are provided. All
combinations of the various complement inhibitors, complement
inhibitor characteristics (e.g., compound class, molecular weight,
half-life, molecular target, etc.), anti-Th17 agents, and dosing
parameters (e.g., dosing interval, route of administration, etc.),
and disorders disclosed herein are contemplated in various
embodiments. All combinations of the various complement inhibitors,
complement inhibitor characteristics (e.g., compound class,
molecular weight, half-life, molecular target, etc.), anti-Th17
agents, anti-Th17 agent characteristics (e.g., compound class,
molecular weight, half-life, molecular target, etc.), and dosing
parameters (e.g., dosing interval, route of administration, etc.),
and disorders disclosed herein are contemplated in various
embodiments.
[0139] In some aspects, the invention provides methods of treating
a chronic complement-mediated disorder or Th17-associated disorder
comprising administering a complement inhibitor and an anti-Th17
agent to a subject in need thereof. In some embodiments the
complement inhibitor and/or anti-Th17 agent are administered
according to any suitable dosing regimen. In some embodiments the
complement inhibitor and anti-Th17 agent are administered according
to a dosing regimen described herein. In some embodiments the
chronic disorder is any chronic complement-mediated disorder or any
Th17-associated disorder. In some aspects, the invention provides
methods of treating a chronic complement-mediated disorder
comprising administering an anti-Th17 agent to a subject in need
thereof. In some embodiments the anti-Th17 agent is administered
according to any suitable dosing regimen. In some embodiments the
anti-Th17 agent is administered according to a dosing regimen
described herein. In some embodiments compositions, e.g.,
pharmaceutical compositions, comprising a complement inhibitor and
an anti-Th17 agent are provided. Exemplary anti-Th17 agents are
discussed in Section V.
III. Complement System
[0140] In order to facilitate understanding of the invention, and
without intending to limit the invention in any way, this section
provides an overview of complement and its pathways of activation.
Further details are found, e.g., in Kuby Immunology, 6.sup.th ed.,
2006; Paul, W. E., Fundamental Immunology, Lippincott Williams
& Wilkins; 6.sup.th ed., 2008; and Walport M J., Complement.
First of two parts. N Engl J Med., 344(14):1058-66, 2001.
[0141] Complement is an arm of the innate immune system that plays
an important role in defending the body against infectious agents.
The complement system comprises more than 30 serum and cellular
proteins that are involved in three major pathways, known as the
classical, alternative, and lectin pathways. The classical pathway
is usually triggered by binding of a complex of antigen and IgM or
IgG antibody to C1 (though certain other activators can also
initiate the pathway). Activated C1 cleaves C4 and C2 to produce
C4a and C4b, in addition to C2a and C2b. C4b and C2a combine to
form C3 convertase, which cleaves C3 to form C3a and C3b. Binding
of C3b to C3 convertase produces C5 convertase, which cleaves C5
into C5a and C5b. C3a, C4a, and C5a are anaphylotoxins and mediate
multiple reactions in the acute inflammatory response. C3a and C5a
are also chemotactic factors that attract immune system cells such
as neutrophils.
[0142] The alternative pathway is initiated by and amplified at,
e.g., microbial surfaces and various complex polysaccharides. In
this pathway, hydrolysis of C3 to C3(H2O), which occurs
spontaneously at a low level, leads to binding of factor B, which
is cleaved by factor D, generating a fluid phase C3 convertase that
activates complement by cleaving C3 into C3a and C3b. C3b binds to
targets such as cell surfaces and forms a complex with factor B,
which is later cleaved by factor D, resulting in a C3 convertase.
Surface-bound C3 convertases cleave and activate additional C3
molecules, resulting in rapid C3b deposition in close proximity to
the site of activation and leading to formation of additional C3
convertase, which in turn generates additional C3b. This process
results in a cycle of C3 cleavage and C3 convertase formation that
signicantly amplifies the response. Cleavage of C3 and binding of
another molecule of C3b to the C3 convertase gives rise to a C5
convertase. C3 and C5 convertases of this pathway are regulated by
host cell molecules CR1, DAF, MCP, CD59, and fH. The mode of action
of these proteins involves either decay accelerating activity
(i.e., ability to dissociate convertases), ability to serve as
cofactors in the degradation of C3b or C4b by factor I, or both.
Normally the presence of complement regulatory proteins on host
cell surfaces prevents significant complement activation from
occurring thereon.
[0143] The C5 convertases produced in both pathways cleave C5 to
produce C5a and C5b. C5b then binds to C6, C7, and C8 to form
C5b-8, which catalyzes polymerization of C9 to form the C5b-9
membrane attack complex (MAC). The MAC inserts itself into target
cell membranes and causes cell lysis. Small amounts of MAC on the
membrane of cells may have a variety of consequences other than
cell death.
[0144] The lectin complement pathway is initiated by binding of
mannose-binding lectin (MBL) and MBL-associated serine protease
(MASP) to carbohydrates. The MB1-1 gene (known as LMAN-1 in humans)
encodes a type I integral membrane protein localized in the
intermediate region between the endoplasmic reticulum and the
Golgi. The MBL-2 gene encodes the soluble mannose-binding protein
found in serum. In the human lectin pathway, MASP-1 and MASP-2 are
involved in the proteolysis of C4 and C2, leading to a C3
convertase described above.
[0145] Complement activity is regulated by various mammalian
proteins referred to as complement control proteins (CCPs) or
regulators of complement activation (RCA) proteins (U.S. Pat. No.
6,897,290). These proteins differ with respect to ligand
specificity and mechanism(s) of complement inhibition. They may
accelerate the normal decay of convertases and/or function as
cofactors for factor I, to enzymatically cleave C3b and/or C4b into
smaller fragments. CCPs are characterized by the presence of
multiple (typically 4-56) homologous motifs known as short
consensus repeats (SCR), complement control protein (CCP) modules,
or SUSHI domains, about 50-70 amino acids in length that contain a
conserved motif including four disulfide-bonded cysteines (two
disulfide bonds), proline, tryptophan, and many hydrophobic
residues. The CCP family includes complement receptor type 1 (CR1;
C3b:C4b receptor), complement receptor type 2 (CR2), membrane
cofactor protein (MCP; CD46), decay-accelerating factor (DAF),
complement factor H (fH), and C4b-binding protein (C4 bp). CD59 is
a membrane-bound complement regulatory protein unrelated
structurally to the CCPs. Complement regulatory proteins normally
serve to limit complement activation that might otherwise occur on
cells and tissues of the mammalian, e.g., human host.
IV. Complement Inhibitors
[0146] General
[0147] A variety of different complement inhibitors may be used in
various embodiments of the invention. In general, a complement
inhibitor can belong to any of a number of compound classes such as
peptides, polypeptides, antibodies, small molecules, and nucleic
acids (e.g., aptamers, RNAi agents such as short interfering RNAs).
In certain embodiments a complement inhibitor inhibits an enzymatic
activity of a complement protein. The enzymatic activity may be
proteolytic activity, such as ability to cleave another complement
protein. In some embodiments, a complement inhibitor inhibits
cleavage of C3, C5, or factor B. In some embodiments, a complement
inhibitor acts on C3. In some embodiments, a complement inhibitor
acts on a complement component that lies upstream of C3 in the
complement activation cascade. In some embodiments, a complement
inhibitor inhibits activation or activity of at least one soluble
complement protein produced in the respiratory system. In certain
embodiments a complement inhibitor that inhibits at least the
classical pathway of complement activation is used. In certain
embodiments a complement inhibitor that inhibits both the classical
and the alternative pathway is used. In some embodiments a
complement inhibitor that inhibits C3 activation or activity is
used. In some embodiments, a complement inhibitor inhibits
activation of at least one complement receptor protein expressed in
the respiratory system. In certain embodiments the complement
receptor protein is a receptor for C3a. In certain embodiments the
complement receptor protein is a receptor for C5a.
[0148] In some embodiments, a complement inhibitor comprises an
antibody that substantially lacks the capacity to activate
complement. For example, the antibody may have less than 10%, less
than 5%, or less than 1% complement stimulating activity as
compared with full length human IgG1. In some embodiments, the
antibody comprises a CH2 domain that has reduced ability to bind
C1q as compared with human IgG1 CH2 domain. In some embodiments,
the antibody contains CH1, CH2, and/or CH3 domains from human IgG4
and/or does not contain CH1, CH2, and/or CH3 domains from human
IgG1.
[0149] In some embodiments, a complement inhibitor used in, e.g.,
an inventive dosing regimen, has a molecular weight of 1 kD or
less. In some embodiments, a complement inhibitor has a molecular
weight between 1 kD and 2 kD, between 2 kD and 5 kD, between 5 kD
and 10 kD, between 10 kD and 20 kD, between 20 kD and 30 kD,
between 30 kD and 50 kD, between 50 kD and 100 kD, or between 100
kD and 200 kD.
[0150] A complement inhibitor may be at least in part identical to
a naturally occurring complement inhibiting agent or a variant or
fragment thereof. A variety of different omplement inhibiting
polypeptides are produced by viruses (e.g., Poxviruses,
Herpesviruses), bacteria (e.g., Staphylococcus), and other
microorganisms. Complement inhibiting proteins are produced by
various parasites, e.g., ectoparasites, such as ticks. A complement
inhibitor can comprise at least a portion of a mammalian complement
control or complement regulatory protein or receptor. See Ricklin,
D., et al. "Complement-targeted Therapeutics", Nature
Biotechnology, 25(11): 1265-75, 2007, for discussion of complement
inhibitors that are or have been in preclinical or clinical
development for various disorders and may be used in various
embodiments of the inventive methods.
[0151] In some embodiments a complement inhibitor comprises an
adnectin, affibody, anticalin, or other type of polypeptide
sometimes used in the art in lieu of an antibody, wherein the
polypeptide binds to a complement component.
[0152] The following sections discuss non-limiting exemplary
complement inhibitors of use in embodiments of the present
invention. Complement inhibitors have been classified in various
groups for purposes of convenience. It will be understood that
certain complement inhibitors fall into multiple categories.
[0153] In some embodiments, a complement inhibitor that binds to
substantially the same binding site (e.g., a binding site on a
complement component such as C3, C5, factor B, factor D, or an
active complement split product) as a complement inhibitor
described herein is used. In general, the ability of first and
second agents to bind to substantially the same site on a target
molecule, such as a complement component or receptor, can be
assessed using methods known in the art, such as competition
assays, molecular modeling, etc. (See, e.g., discussion of
compstatin analog mimetics.) Optionally the first and/or second
agent can be labeled with a detectable label, e.g., a radiolabel,
fluorescent label, etc. Optionally the target molecule, first
agent, or second agent is immobilized on a support, e.g., a slide,
filter, chip, beads, etc. In some embodiments, a second antibody
that binds to substantially the same binding site as a first
antibody comprises one or more CDR(s) that are at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to CDR(s)
of the first antibody.
[0154] Compounds that Inhibit C3 Activation or Activity
[0155] Compstatin Analogs and Mimetics
[0156] Compstatin is a cyclic peptide that binds to C3 and inhibits
complement activation by, e.g., inhibiting cleavage of C3 to C3a
and C3b by convertase. U.S. Pat. No. 6,319,897 describes a peptide
having the sequence
Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys]-Thr (SEQ ID NO:
1), with the disulfide bond between the two cysteines denoted by
brackets. It will be understood that the name "compstatin" was not
used in U.S. Pat. No. 6,319,897 but was subsequently adopted in the
scientific and patent literature (see, e.g., Morikis, et al.,
Protein Sci., 7(3):619-27, 1998) to refer to a peptide having the
same sequence as SEQ ID NO: 2 disclosed in U.S. Pat. No. 6,319,897,
but amidated at the C terminus as shown in Table 2 (SEQ ID NO: 8).
The term "compstatin" is used herein consistently with such usage
(i.e., to refer to SEQ ID NO: 8). Compstatin analogs that have
higher complement inhibiting activity than compstatin have been
developed. See, e.g., WO2004/026328 (PCT/US2003/029653), Morikis,
D., et al., Biochem Soc Trans. 32(Pt 1):28-32, 2004, Mallik, B., et
al., J. Med. Chem., 274-286, 2005; Katragadda, M., et al. J. Med.
Chem., 49: 4616-4622, 2006; WO2007062249 (PCT/US2006/045539);
WO2007044668 (PCT/US2006/039397), WO/2009/046198
(PCT/US2008/078593); WO/2010/127336 (PCT/US2010/033345) and
discussion below.
[0157] Compstatin analogs may be acetylated or amidated, e.g., at
the N-terminus and/or C-terminus For example, compstatin analogs
may be acetylated at the N-terminus and amidated at the C-terminus
Consistent with usage in the art, "compstatin" as used herein, and
the activities of compstatin analogs described herein relative to
that of compstatin, refer to compstatin amidated at the C-terminus
(Mallik, 2005, supra).
[0158] Concatamers or multimers of compstatin or a complement
inhibiting analog thereof are also of use in the present
invention.
[0159] As used herein, the term "compstatin analog" includes
compstatin and any complement inhibiting analog thereof. The term
"compstatin analog" encompasses compstatin and other compounds
designed or identified based on compstatin and whose complement
inhibiting activity is at least 50% as great as that of compstatin
as measured, e.g., using any complement activation assay accepted
in the art or substantially similar or equivalent assays. Certain
suitable assays are described in U.S. Pat. No. 6,319,897,
WO2004/026328, Morikis, supra, Mallik, supra, Katragadda 2006,
supra, WO2007062249 (PCT/US2006/045539); WO2007044668
(PCT/US2006/039397), WO/2009/046198 (PCT/US2008/078593); and/or
WO/2010/127336 (PCT/US2010/033345). The assay may, for example,
measure alternative or classical pathway-mediated erythrocyte lysis
or be an ELISA assay. In some embodiments, an assay described in
WO/2010/135717 (PCT/US2010/035871) is used.
[0160] The activity of a compstatin analog may be expressed in
terms of its IC.sub.50 (the concentration of the compound that
inhibits complement activation by 50%), with a lower IC.sub.50
indicating a higher activity as recognized in the art. The activity
of a preferred compstatin analog for use in the present invention
is at least as great as that of compstatin. It is noted that
certain modifications known to reduce or eliminate complement
inhibiting activity and may be explicitly excluded from any
embodiment of the invention. The IC.sub.50 of compstatin has been
measured as 12 .mu.M using an alternative pathway-mediated
erythrocyte lysis assay (WO2004/026328). It will be appreciated
that the precise IC.sub.50 value measured for a given compstatin
analog will vary with experimental conditions (e.g., the serum
concentration used in the assay). Comparative values, e.g.,
obtained from experiments in which IC.sub.50 is determined for
multiple different compounds under substantially identical
conditions, are of use. In one embodiment, the IC.sub.50 of the
compstatin analog is no more than the IC.sub.50 of compstatin. In
certain embodiments of the invention the activity of the compstatin
analog is between 2 and 99 times that of compstatin (i.e., the
analog has an IC.sub.50 that is less than the IC.sub.50 of
compstatin by a factor of between 2 and 99). For example, the
activity may be between 10 and 50 times as great as that of
compstatin, or between 50 and 99 times as great as that of
compstatin. In certain embodiments of the invention the activity of
the compstatin analog is between 99 and 264 times that of
compstatin. For example, the activity may be 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or
264 times as great as that of compstatin. In certain embodiments
the activity is between 250 and 300, 300 and 350, 350 and 400, or
400 and 500 times as great as that of compstatin. The invention
further contemplates compstatin analogs having activities between
500 and 1000 times that of compstatin, or more, e.g., between 1000
and 2000 times that of compstatin, or more. In certain embodiments
the IC.sub.50 of the compstatin analog is between about 0.2 .mu.M
and about 0.5 .mu.M. In certain embodiments the IC.sub.50 of the
compstatin analog is between about 0.1 .mu.M and about 0.2 .mu.M.
In certain embodiments the IC.sub.50 of the compstatin analog is
between about 0.05 .mu.M and about 0.1 .mu.M. In certain
embodiments the IC.sub.50 of the compstatin analog is between about
0.001 .mu.M and about 0.05 .mu.M.
[0161] The K.sub.d of compstatin binding to C3 can be measured
using isothermal titration calorimetry (Katragadda, et al., J.
Biol. Chem., 279(53), 54987-54995, 2004). Binding affinity of a
variety of compstatin analogs for C3 has been correlated with their
activity, with a lower K.sub.d indicating a higher binding
affinity, as recognized in the art. A linear correlation between
binding affinity and activity was shown for certain analogs tested
(Katragadda, 2004, supra; Katragadda 2006, supra). In certain
embodiments of the invention the compstatin analog binds to C3 with
a K.sub.d of between 0.1 .mu.M and 1.0 .mu.M, between 0.05 .mu.M
and 0.1 .mu.M, between 0.025 .mu.M and 0.05 .mu.M, between 0.015
.mu.M and 0.025 .mu.M, between 0.01 .mu.M and 0.015 .mu.M, or
between 0.001 .mu.M and 0.01 .mu.M.
[0162] Compounds "designed or identified based on compstatin"
include, but are not limited to, compounds that comprise an amino
acid chain whose sequence is obtained by (i) modifying the sequence
of compstatin (e.g., replacing one or more amino acids of the
sequence of compstatin with a different amino acid or amino acid
analog, inserting one or more amino acids or amino acid analogs
into the sequence of compstatin, or deleting one or more amino
acids from the sequence of compstatin); (ii) selection from a phage
display peptide library in which one or more amino acids of
compstatin is randomized, and optionally further modified according
to method (i); or (iii) identified by screening for compounds that
compete with compstatin or any analog thereof obtained by methods
(i) or (ii) for binding to C3 or a fragment thereof. Many useful
compstatin analogs comprise a hydrophobic cluster, a .beta.-turn,
and a disulfide bridge.
[0163] In certain embodiments of the invention the sequence of the
compstatin analog comprises or consists essentially of a sequence
that is obtained by making 1, 2, 3, or 4 substitutions in the
sequence of compstatin, i.e., 1, 2, 3, or 4 amino acids in the
sequence of compstatin is replaced by a different standard amino
acid or by a non-standard amino acid. In certain embodiments of the
invention the amino acid at position 4 is altered. In certain
embodiments of the invention the amino acid at position 9 is
altered. In certain embodiments of the invention the amino acids at
positions 4 and 9 are altered. In certain embodiments of the
invention only the amino acids at positions 4 and 9 are altered. In
certain embodiments of the invention the amino acid at position 4
or 9 is altered, or in certain embodiments both amino acids 4 and 9
are altered, and in addition up to 2 amino acids located at
positions selected from 1, 7, 10, 11, and 13 are altered. In
certain embodiments of the invention the amino acids at positions
4, 7, and 9 are altered. In certain embodiments of the invention
amino acids at position 2, 12, or both are altered, provided that
the alteration preserves the ability of the compound to be
cyclized. Such alteration(s) at positions 2 and/or 12 may be in
addition to the alteration(s) at position 1, 4, 7, 9, 10, 11,
and/or 13. Optionally the sequence of any of the compstatin analogs
whose sequence is obtained by replacing one or more amino acids of
compstatin sequence further includes up to 1, 2, or 3 additional
amino acids at the C-terminus. In one embodiment, the additional
amino acid is Gly. Optionally the sequence of any of the compstatin
analogs whose sequence is obtained by replacing one or more amino
acids of compstatin sequence further includes up to 5, or up to 10
additional amino acids at the C-terminus. It should be understood
that compstatin analogs may have any one or more of the
characteristics or features of the various embodiments described
herein, and characteristics or features of any embodiment may
additionally characterize any other embodiment described herein,
unless otherwise stated or evident from the context. In certain
embodiments of the invention the sequence of the compstatin analog
comprises or consists essentially of a sequence identical to that
of compstatin except at positions corresponding to positions 4 and
9 in the sequence of compstatin.
[0164] Compstatin and certain compstatin analogs having somewhat
greater activity than compstatin contain only standard amino acids
("standard amino acids" are glycine, leucine, isoleucine, valine,
alanine, phenylalanine, tyrosine, tryptophan, aspartic acid,
asparagine, glutamic acid, glutamine, cysteine, methionine,
arginine, lysine, proline, serine, threonine and histidine).
Certain compstatin analogs having improved activity incorporate one
or more non-standard amino acids. Useful non-standard amino acids
include singly and multiply halogenated (e.g., fluorinated) amino
acids, D-amino acids, homo-amino acids, N-alkyl amino acids,
dehydroamino acids, aromatic amino acids (other than phenylalanine,
tyrosine and tryptophan), ortho-, meta- or para-aminobenzoic acid,
phospho-amino acids, methoxylated amino acids, and
.alpha.,.alpha.-disubstituted amino acids. In certain embodiments
of the invention, a compstatin analog is designed by replacing one
or more L-amino acids in a compstatin analog described elsewhere
herein with the corresponding D-amino acid. Such compounds and
methods of use thereof are an aspect of the invention. Exemplary
non-standard amino acids of use include 2-naphthylalanine (2-NaI),
1-naphthylalanine (1-NaI), 2-indanylglycine carboxylic acid (2Ig1),
dihydrotrpytophan (Dht), 4-benzoyl-L-phenylalanine (Bpa),
2-.alpha.-aminobutyric acid (2-Abu), 3-.alpha.-aminobutyric acid
(3-Abu), 4-.alpha.-aminobutyric acid (4-Abu), cyclohexylalanine
(Cha), homocyclohexylalanine (hCha), 4-fluoro-L-tryptophan (4fW),
5-fluoro-L-tryptophan (5fW), 6-fluoro-L-tryptophan (6fW),
4-hydroxy-L-tryptophan (4OH--W), 5-hydroxy-L-tryptophan (5OH--W),
6-hydroxy-L-tryptophan (6OH--W), 1-methyl-L-tryptophan (1MeW),
4-methyl-L-tryptophan (4MeW), 5-methyl-L-tryptophan (5MeW),
7-aza-L-tryptophan (7aW), .alpha.-methyl-L-tryptophan (.alpha.MeW),
.beta.-methyl-L-tryptophan (.beta.MeW), N-methyl-L-tryptophan
(NMeW), ornithine (orn), citrulline, norleucine, .gamma.-glutamic
acid, etc.
[0165] In certain embodiments of the invention the compstatin
analog comprises one or more Trp analogs (e.g., at position 4
and/or 7 relative to the sequence of compstatin). Exemplary Trp
analogs are mentioned above. See also Beene, et. al. Biochemistry
41: 10262-10269, 2002 (describing, inter alia, singly- and
multiply-halogenated Trp analogs); Babitzke & Yanofsky, J.
Biol. Chem. 270: 12452-12456, 1995 (describing, inter alia,
methylated and halogenated Trp and other Trp and indole analogs);
and U.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097,
4,576,750 and 4,299,838. Other Trp analogs include variants that
are substituted (e.g., by a methyl group) at the .alpha. or .beta.
carbon and, optionally, also at one or more positions of the indole
ring Amino acids comprising two or more aromatic rings, including
substituted, unsubstituted, or alternatively substituted variants
thereof, are of interest as Trp analogs. In certain embodiments of
the invention the Trp analog, e.g., at position 4, is 5-methoxy,
5-methyl-, 1-methyl-, or 1-formyl-tryptophan. In certain
embodiments of the invention a Trp analog (e.g., at position 4)
comprising a 1-alkyl substituent, e.g., a lower alkyl (e.g.,
C.sub.1-C.sub.5) substituent is used. In certain embodiments,
N(.alpha.) methyl tryptophan or 5-methyltryptophan is used. In some
embodiments, an analog comprising a 1-alkanyol substituent, e.g., a
lower alkanoyl (e.g., C.sub.1-C.sub.5) is used. Examples include
1-acetyl-L-tryptophan and L-.beta.-tryptophan.
[0166] In certain embodiments the Trp analog has increased
hydrophobic character relative to Trp. For example, the indole ring
may be substituted by one or more alkyl (e.g., methyl) groups. In
certain embodiments the Trp analog participates in a hydrophobic
interaction with C3. Such a Trp analog may be located, e.g., at
position 4 relative to the sequence of compstatin. In certain
embodiments the Trp analog comprises a substituted or unsubstituted
bicyclic aromatic ring component or two or more substituted or
unsubstituted monocyclic aromatic ring components.
[0167] In certain embodiments the Trp analog has increased
propensity to form hydrogen bonds with C3 relative to Trp but does
not have increased hydrophobic character relative to Trp. The Trp
analog may have increased polarity relative to Trp and/or an
increased ability to participate in an electrostatic interaction
with a hydrogen bond donor on C3. Certain exemplary Trp analogs
with an increased hydrogen bond forming character comprise an
electronegative substituent on the indole ring. Such a Trp analog
may be located, e.g., at position 7 relative to the sequence of
compstatin.
[0168] In certain embodiments of the invention the compstatin
analog comprises one or more Ala analogs (e.g., at position 9
relative to the sequence of compstatin), e.g., Ala analogs that are
identical to Ala except that they include one or more CH.sub.2
groups in the side chain. In certain embodiments the Ala analog is
an unbranched single methyl amino acid such as 2-Abu. In certain
embodiments of the invention the compstatin analog comprises one or
more Trp analogs (e.g., at position 4 and/or 7 relative to the
sequence of compstatin) and an Ala analog (e.g., at position 9
relative to the sequence of compstatin).
[0169] In certain embodiments of the invention the compstatin
analog is a compound that comprises a peptide that has a sequence
of (X'aa).sub.n-Gln-Asp-Xaa-Gly-(X''aa).sub.m, (SEQ ID NO: 2)
wherein each X'aa and each X''aa is an independently selected amino
acid or amino acid analog, wherein Xaa is Trp or an analog of Trp,
and wherein n>1 and m>1 and n+m is between 5 and 21. The
peptide has a core sequence of Gln-Asp-Xaa-Gly (SEQ ID NO: 71),
where Xaa is Trp or an analog of Trp, e.g., an analog of Trp having
increased propensity to form hydrogen bonds with an H-bond donor
relative to Trp but, in certain embodiments, not having increased
hydrophobic character relative to Trp. For example, the analog may
be one in which the indole ring of Trp is substituted with an
electronegative moiety, e.g., a halogen such as fluorine. In one
embodiment Xaa is 5-fluorotryptophan. Absent evidence to the
contrary, one of skill in the art would recognize that any
non-naturally occurring peptide whose sequence comprises this core
sequence and that inhibits complement activation and/or binds to C3
will have been designed based on the sequence of compstatin. In an
alternative embodiment Xaa is an amino acid or amino acid analog
other than a Trp analog that allows the Gln-Asp-Xaa-Gly (SEQ ID NO:
71) peptide to form a .beta.-turn.
[0170] In certain embodiments of the invention the peptide has a
core sequence of X'aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X'aa
and Xaa are selected from Trp and analogs of Trp. In certain
embodiments of the invention the peptide has a core sequence of
X'aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X'aa and Xaa are
selected from Trp, analogs of Trp, and other amino acids or amino
acid analogs comprising at least one aromatic ring. In certain
embodiments of the invention the core sequence forms a .beta.-turn
in the context of the peptide. The .beta.-turn may be flexible,
allowing the peptide to assume two or more conformations as
assessed for example, using nuclear magnetic resonance (NMR). In
certain embodiments X'aa is an analog of Trp that comprises a
substituted or unsubstituted bicyclic aromatic ring component or
two or more substituted or unsubstituted monocyclic aromatic ring
components. In certain embodiments of the invention X'aa is
selected from the group consisting of 2-napthylalanine,
1-napthylalanine, 2-indanylglycine carboxylic acid,
dihydrotryptophan, and benzoylphenylalanine. In certain embodiments
of the invention X'aa is an analog of Trp that has increased
hydrophobic character relative to Trp. For example, X'aa may be
1-methyltryptophan. In certain embodiments of the invention Xaa is
an analog of Trp that has increased propensity to form hydrogen
bonds relative to Trp but, in certain embodiments, not having
increased hydrophobic character relative to Trp. In certain
embodiments of the invention the analog of Trp that has increased
propensity to form hydrogen bonds relative to Trp comprises a
modification on the indole ring of Trp, e.g., at position 5, such
as a substitution of a halogen atom for an H atom at position 5.
For example, Xaa may be 5-fluorotryptophan.
[0171] In certain embodiments of the invention the peptide has a
core sequence of X'aa-Gln-Asp-Xaa-Gly-X''aa (SEQ ID NO: 4), where
X'aa and Xaa are each independently selected from Trp and analogs
of Trp and X''aa is selected from His, Ala, analogs of Ala, Phe,
and Trp. In certain embodiments of the invention X'aa is an analog
of Trp that has increased hydrophobic character relative to Trp,
such as 1-methyltryptophan or another Trp analog having an alkyl
substituent on the indole ring (e.g., at position 1, 4, 5, or 6).
In certain embodiments X'aa is an analog of Trp that comprises a
substituted or unsubstituted bicyclic aromatic ring component or
two or more substituted or unsubstituted monocyclic aromatic ring
components. In certain embodiments of the invention X'aa is
selected from the group consisting of 2-napthylalanine,
1-napthylalanine, 2-indanylglycine carboxylic acid,
dihydrotryptophan, and benzoylphenylalanine. In certain embodiments
of the invention Xaa is an analog of Trp that has increased
propensity to form hydrogen bonds with C3 relative to Trp but, in
certain embodiments, not having increased hydrophobic character
relative to Trp. In certain embodiments of the invention the analog
of Trp that has increased propensity to form hydrogen bonds
relative to Trp comprises a modification on the indole ring of Trp,
e.g., at position 5, such as a substitution of a halogen atom for
an H atom at position 5. For example, Xaa may be
5-fluorotryptophan. In certain embodiments X''aa is Ala or an
analog of Ala such as Abu or another unbranched single methyl amino
acid. In certain embodiments of the invention the peptide has a
core sequence of X'aa-Gln-Asp-Xaa-Gly-X''aa (SEQ ID NO: 4), where
X'aa and Xaa are each independently selected from Trp, analogs of
Trp, and amino acids or amino acid analogs comprising at least one
aromatic side chain, and X''aa is selected from His, Ala, analogs
of Ala, Phe, and Trp. In certain embodiments X''aa is selected from
analogs of Trp, aromatic amino acids, and aromatic amino acid
analogs.
[0172] In certain preferred embodiments of the invention the
peptide is cyclic. The peptide may be cyclized via a bond between
any two amino acids, one of which is (X'aa). and the other of which
is located within (X''aa).sub.m. In certain embodiments the cyclic
portion of the peptide is between 9 and 15 amino acids in length,
e.g., 10-12 amino acids in length. In certain embodiments the
cyclic portion of the peptide is 11 amino acids in length, with a
bond (e.g., a disulfide bond) between amino acids at positions 2
and 12. For example, the peptide may be 13 amino acids long, with a
bond between amino acids at positions 2 and 12 resulting in a
cyclic portion 11 amino acids in length.
[0173] In certain embodiments the peptide comprises or consists of
the sequence
X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X''aa1-X''aa2-X''aa3-X''-
aa4-X''aa5 (SEQ ID NO: 5). In certain embodiments X'aa4 and Xaa are
selected from Trp and analogs of Trp, and X'aa1, X'aa2, X'aa3,
X''aa1, X''aa2, X''aa3, X''aa4, and X''aa5 are independently
selected from among amino acids and amino acid analogs. In certain
embodiments X'aa4 and Xaa are selected from aromatic amino acids
and aromatic amino acid analogs. Any one or more of X'aa1, X'aa2,
X'aa3, X''aa1, X''aa2, X''aa3, X''aa4, and X''aa5 may be identical
to the amino acid at the corresponding position in compstatin. In
one embodiment, X''aa1 is Ala or a single methyl unbranched amino
acid. The peptide may be cyclized via a covalent bond between (i)
X'aa1, X'aa2, or X'aa3; and (ii) X''aa2, X''aa3, X''aa4 or X''aa5.
In one embodiment the peptide is cyclized via a covalent bond
between X'aa2 and X''aa4. In one embodiment the covalently bound
amino acid are each Cys and the covalent bond is a disulfide (S--S)
bond. In other embodiments the covalent bond is a C--C, C--O, C--S,
or C--N bond. In certain embodiments one of the covalently bound
residues is an amino acid or amino acid analog having a side chain
that comprises a primary or secondary amine, the other covalently
bound residue is an amino acid or amino acid analog having a side
chain that comprises a carboxylic acid group, and the covalent bond
is an amide bond Amino acids or amino acid analogs having a side
chain that comprises a primary or secondary amine include lysine
and diaminocarboxylic acids of general structure
NH.sub.2(CH.sub.2).sub.nCH(NH.sub.2)COOH such as
2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid (daba),
and ornithine (orn), wherein n=1 (dapa), 2 (daba), and 3 (orn),
respectively. Examples of amino acids having a side chain that
comprises a carboxylic acid group include dicarboxylic amino acids
such as glutamic acid and aspartic acid. Analogs such as
beta-hydroxy-L-glutamic acid may also be used. In some embodiments
a peptide is cyclized with a thioether bond, e.g., as described in
PCT/US2011/052442 (WO/2012/040259). For example, in some
embodiments a disulfide bond in any of the peptides is replaced
with a thioether bond. In some embodiments, a cystathionine is
formed. In some embodiments the cystathionine is a
delta-cystathionine or a gamma-cystathionine. In some embodiments a
modification comprises replacement of a Cys-Cys disulfide bond
between cysteines at X'aa2 and X''aa4 in SEQ ID NO: 5 (or
corresponding positions in other sequences) with addition of a
CH.sub.2, to form a homocysteine at X'aa2 or X''aa4, and
introduction of a thioether bond, to form a cystathionine. In one
embodiment, the cystathionine is a gamma-cystathionine. In another
embodiment, the cystathionine is a delta-cystathionine. Another
modification of use in certain embodiments comprises replacement of
the disulfide bond with a thioether bond without the addition of a
CH.sub.2, thereby forming a lantithionine. In some embodiments a
compstatin analog having a thioether in place of a disulfide bond
has increased stability, at least under some conditions, as
compared with the compstatin analog having the disulfide bond.
[0174] In certain embodiments, the compstatin analog is a compound
that comprises a peptide having a sequence:
[0175] Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4
(SEQ ID NO: 6); wherein:
Xaa1 is Ile, Val, Leu, B.sup.1-Ile, B.sup.1-Val, B.sup.1-Leu or a
dipeptide comprising Gly-Ile or B.sup.1-Gly-Ile, and B.sup.1
represents a first blocking moiety; Xaa2 and Xaa2* are
independently selected from Trp and analogs of Trp; Xaa3 is His,
Ala or an analog of Ala, Phe, Trp, or an analog of Trp; Xaa4 is
L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Ala and
Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxy
terminal --OH of any of the L-Thr, D-Thr, Ile, Val, Gly, Ala, or
Asn optionally is replaced by a second blocking moiety B.sup.2; and
the two Cys residues are joined by a disulfide bond. In some
embodiments, Xaa4 is Leu, Nle, His, or Phe or a depeptide selected
from Xaa5-Ala and Xaa5-Asn, or a tripeptide Xaa5-Ala-Asn, wherein
Xaa5 is selected from Leu, Nle, His or Phe, and wherein a carboxy
terminal --OH of any of the L-Thr, D-Thr, Ile, Val, Gly, Leu, Nle,
His, Phe, Ala, or Asn optionally is replaced by a second blocking
moiety B.sup.2; and the two Cys residues are joined by a disulfide
bond.
[0176] In other embodiments Xaa1 is absent or is any amino acid or
amino acid analog, and Xaa2, Xaa2*, Xaa3, and Xaa4 are as defined
above. If Xaa1 is absent, the N-terminal Cys residue may have a
blocking moiety B.sup.1 attached thereto.
[0177] In another embodiment, Xaa4 is any amino acid or amino acid
analog and Xaa1, Xaa2, Xaa2*, and Xaa3 are as defined above. In
another embodiment Xaa4 is a dipeptide selected from the group
consisting of: Thr-Ala and Thr-Asn, wherein the carboxy terminal
--OH or the Ala or Asn is optionally replaced by a second blocking
moiety B.sup.2.
[0178] In any of the embodiments of the compstatin analog of SEQ ID
NO: 6, Xaa2 may be Trp.
[0179] In any of the embodiments of the compstatin analog of SEQ ID
NO: 6, Xaa2 may be an analog of Trp comprising a substituted or
unsubstituted bicyclic aromatic ring component or two or more
substituted or unsubstituted monocyclic aromatic ring components.
For example, the analog of Trp may be selected from
2-naphthylalanine (2-NaI), 1-naphthylalanine (1-NaI),
2-indanylglycine carboxylic acid (Ig1), dihydrotrpytophan (Dht),
and 4-benzoyl-L-phenylalanine.
[0180] In any of the embodiments of the compstatin analog of SEQ ID
NO: 6, Xaa2 may be an analog of Trp having increased hydrophobic
character relative to Trp. For example, the analog of Trp may be
selected from 1-methyltryptophan, 4-methyltryptophan,
5-methyltryptophan, and 6-methyltryptophan. In one embodiment, the
analog of Trp is 1-methyltryptophan. In one embodiment, Xaa2 is
1-methyltryptophan, Xaa2* is Trp, Xaa3 is Ala, and the other amino
acids are identical to those of compstatin.
[0181] In any of the embodiments of the compstatin analog of SEQ ID
NO: 6, Xaa2* may be an analog of Trp such as an analog of Trp
having increased hydrogen bond forming propensity with C3 relative
to Trp, which, in certain embodiments, does not have increased
hydrophobic character relative to Trp. In certain embodiments the
analog of Trp comprises an electronegative substituent on the
indole ring. For example, the analog of Trp may be selected from
5-fluorotryptophan and 6-fluorotryptophan.
[0182] In certain embodiments of the invention Xaa2 is Trp and
Xaa2* is an analog of Trp having increased hydrogen bond forming
propensity with C3 relative to Trp which, in certain embodiments,
does not have increased hydrophobic character relative to Trp. In
certain embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2
is analog of Trp having increased hydrophobic character relative to
Trp such as an analog of Trp selected from 1-methyltryptophan,
4-methyltryptophan, 5-methyltryptophan, and 6-methyltryptophan, and
Xaa2* is an analog of Trp having increased hydrogen bond forming
propensity with C3 relative to Trp which, in certain embodiments,
does not have increased hydrophobic character relative to Trp. For
example, in one embodiment Xaa2 is methyltryptophan and Xaa2* is
5-fluorotryptophan.
[0183] In certain of the afore-mentioned embodiments, Xaa3 is Ala.
In certain of the afore-mentioned embodiments Xaa3 is a single
methyl unbranched amino acid, e.g., Abu.
[0184] The invention further provides compstatin analogs of SEQ ID
NO: 6, as described above, wherein Xaa2 and Xaa2* are independently
selected from Trp, analogs of Trp, and other amino acids or amino
acid analogs that comprise at least one aromatic ring, and Xaa3 is
His, Ala or an analog of Ala, Phe, Trp, an analog of Trp, or
another aromatic amino acid or aromatic amino acid analog.
[0185] In certain embodiments of the invention the blocking moiety
present at the N- or C-terminus of any of the compstatin analogs
described herein is any moiety that stabilizes a peptide against
degradation that would otherwise occur in mammalian (e.g., human or
non-human primate) blood or interstitial fluid. For example,
blocking moiety B.sup.1 could be any moiety that alters the
structure of the N-terminus of a peptide so as to inhibit cleavage
of a peptide bond between the N-terminal amino acid of the peptide
and the adjacent amino acid. Blocking moiety B.sup.2 could be any
moiety that alters the structure of the C-terminus of a peptide so
as to inhibit cleavage of a peptide bond between the C-terminal
amino acid of the peptide and the adjacent amino acid. Any suitable
blocking moieties known in the art could be used. In certain
embodiments of the invention blocking moiety B.sup.1 comprises an
acyl group (i.e., the portion of a carboxylic acid that remains
following removal of the --OH group). The acyl group typically
comprises between 1 and 12 carbons, e.g., between 1 and 6 carbons.
For example, in certain embodiments of the invention blocking
moiety B.sup.1 is selected from the group consisting of: formyl,
acetyl, proprionyl, butyryl, isobutyryl, valeryl, isovaleryl, etc.
In one embodiment, the blocking moiety B.sup.1 is an acetyl group,
i.e., Xaa1 is Ac-Ile, Ac-Val, Ac-Leu, or Ac-Gly-Ile.
[0186] In certain embodiments of the invention blocking moiety
B.sup.2 is a primary or secondary amine (--NH.sub.2 or --NHR.sup.1,
wherein R is an organic moiety such as an alkyl group).
[0187] In certain embodiments of the invention blocking moiety
B.sup.1 is any moiety that neutralizes or reduces the positive
charge that may otherwise be present at the N-terminus at
physiological pH. In certain embodiments of the invention blocking
moiety B.sup.2 is any moiety that neutralizes or reduces the
negative charge that may otherwise be present at the C-terminus at
physiological pH.
[0188] In certain embodiments of the invention, the compstatin
analog is acetylated or amidated at the N-terminus and/or
C-terminus, respectively. A compstatin analog may be acetylated at
the N-terminus, amidated at the C-terminus, and or both acetylated
at the N-terminus and amidated at the C-terminus. In certain
embodiments of the invention a compstatin analog comprises an alkyl
or aryl group at the N-terminus rather than an acetyl group.
[0189] In certain embodiments, the compstatin analog is a compound
that comprises a peptide having a sequence:
[0190] Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4
(SEQ ID NO: 7); wherein:
Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide
comprising Gly-Ile or Ac-Gly-Ile; Xaa2 and Xaa2* are independently
selected from Trp and analogs of Trp; Xaa3 is His, Ala or an analog
of Ala, Phe, Trp, or an analog of Trp; Xaa4 is L-Thr, D-Thr, Ile,
Val, Gly, a dipeptide selected from Thr-Ala and Thr-Asn, or a
tripeptide comprising Thr-Ala-Asn, wherein a carboxy terminal --OH
of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn optionally is
replaced by --NH.sub.2; and the two Cys residues are joined by a
disulfide bond. In some embodiments, Xaa4 is Leu, Nle, His, or Phe
or a depeptide selected from Xaa5-Ala and Xaa5-Asn, or a tripeptide
Xaa5-Ala-Asn, wherein Xaa5 is selected from Leu, Nle, His or Phe,
and wherein a carboxy terminal --OH of any of the L-Thr, D-Thr,
Ile, Val, Gly, Leu, Nle, His, Phe, Ala, or Asn optionally is
replaced by a second blocking moiety B2; and the two Cys residues
are joined by a disulfide bond.
[0191] In some embodiments, Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are
as described above for the various embodiments of SEQ ID NO: 6. For
example, in certain embodiments Xaa2* is Trp. In certain
embodiments Xaa2 is an analog of Trp having increased hydrophobic
character relative to Trp, e.g., 1-methyltryptophan. In certain
embodiments Xaa3 is Ala. In certain embodiments Xaa3 is a single
methyl unbranched amino acid.
[0192] In certain embodiments of the invention Xaa1 is Ile and Xaa4
is L-Thr.
[0193] In certain embodiments of the invention Xaa1 is Ile, Xaa2*
is Trp, and Xaa4 is L-Thr.
[0194] The invention further provides compstatin analogs of SEQ ID
NO: 7, as described above, wherein Xaa2 and Xaa2* are independently
selected from Trp, analogs of Trp, other amino acids or aromatic
amino acid analogs, and Xaa3 is His, Ala or an analog of Ala, Phe,
Trp, an analog of Trp, or another aromatic amino acid or aromatic
amino acid analog.
[0195] In certain embodiments of any of the compstatin analogs
described herein, an analog of Phe is used rather than Phe.
[0196] Table 2 provides a non-limiting list of compstatin analogs
useful in the present invention. The analogs are referred to in
abbreviated form in the left column by indicating specific
modifications at designated positions (1-13) as compared to the
parent peptide, compstatin. Consistent with usage in the art,
"compstatin" as used herein, and the activities of compstatin
analogs described herein relative to that of compstatin, refer to
the compstatin peptide amidated at the C-terminus Unless otherwise
indicated, peptides in Table 2 are amidated at the C-terminus Bold
text is used to indicate certain modifications. Activity relative
to compstatin is based on published data and assays described
therein (WO2004/026328, WO2007044668, Mallik, 2005; Katragadda,
2006). Where multiple publications reporting an activity were
consulted, the more recently published value is used, and it will
be recognized that values may be adjusted in the case of
differences between assays. It will also be appreciated that in
certain embodiments of the invention the peptides listed in Table 2
are cyclized via a disulfide bond between the two Cys residues when
used in the therapeutic compositions and methods of the invention.
Alternate means for cyclizing the peptides are also within the
scope of the invention. As noted above, in various embodiments of
the invention one or more amino acid(s) of a compstatin analog
(e.g., any of the compstatin analogs disclosed herein) can be an
N-alkyl amino acid (e.g., an N-methyl amino acid). For example, and
without limitation, at least one amino acid within the cyclic
portion of the peptide, at least one amino acid N-terminal to the
cyclic portion, and/or at least one amino acid C-terminal to the
cyclic portion may be an N-alkyl amino acid, e.g., an N-methyl
amino acid. In some embodiments of the invention, for example, a
compstatin analog comprises an N-methyl glycine, e.g., at the
position corresponding to position 8 of compstatin and/or at the
position corresponding to position 13 of compstatin. In some
embodiments, one or more of the compstatin analogs in Table 2
contains at least one N-methyl glycine, e.g., at the position
corresponding to position 8 of compstatin and/or at the position
corresponding to position 13 of compstatin. In some embodiments,
one or more of the compstatin analogs in contains at least one
N-methyl isoleucine, e.g., at the position corresponding to
position 13 of compstatin. For example, a Thr at or near the
C-terminal end of a peptide whose sequence is listed in Table 2 may
be replaced by N-methyl Ile. As will be appreciated, in some
embodiments the N-methylated amino acids comprise N-methyl Gly at
position 8 and N-methyl Ile at position 13. In some embodiments the
N-methylated amino acids comprise N-methyl Gly in a core sequence
such as SEQ ID NO: 3 or SEQ ID NO: 4.
TABLE-US-00002 TABLE 2 SEQ ID Activity over Peptide Sequence NO:
compstatin Compstatin H-ICVVQDWGHHRCT-CONH2 8 * Ac-compstatin
Ac-ICVVQDWGHHRCT-CONH2 9 3 .times. more Ac-V4Y/H9A
Ac-ICVYQDWGAHRCT-CONH2 10 14 .times. more Ac-V4W/H9A -OH
Ac-ICVWQDWGAHRCT-COOH 11 27 .times. more Ac-V4W/H9A
Ac-ICVWQDWGAHRCT-CONH2 12 45 .times. more Ac-V4W/H9A/T13dT -OH
Ac-ICVWQDWGAHRCdT-COOH 13 55 .times. more Ac-V4(2-Nal)/H9A
Ac-ICV(2-Nal)QDWGAHRCT-CONH2 14 99 .times. more Ac V4(2-Nal)/H9A
-OH Ac-ICV(2-Nal)QDWGAHRCT-COOH 15 38 .times. more Ac V4(1-Nal)/H9A
-OH Ac-ICV(1-Nal)QDWGAHRCT-COOH 16 30 .times. more Ac-V42Igl/H9A
Ac-ICV(2-Igl)QDWGAHRCT-CONH2 17 39 .times. more Ac-V42Igl/H9A -OH
Ac-ICV(2-Igl)QDWGAHRCT-COOH 18 37 .times. more Ac-V4Dht/H9A -OH
Ac-ICVDhtQDWGAHRCT-COOH 19 5 .times. more Ac-V4(Bpa)/H9A -OH
Ac-ICV(Bpa)QDWGAHRCT-COOH 20 49 .times. more Ac-V4(Bpa)/H9A
Ac-ICV(Bpa)QDWGAHRCT-CONH2 21 86 .times. more Ac-V4(Bta)/H9A -OH
Ac-ICV(Bta)QDWGAHRCT-COOH 22 65 .times. more Ac-V4(Bta)/H9A
Ac-ICV(Bta)QDWGAHRCT-CONH2 23 64 .times. more Ac-V4W/H9(2-Abu)
Ac-ICVWQDWG(2-Abu)HRCT-CONH2 24 64 .times. more +GN4W/H9A + AN -OH
H-GICVWQDWGAHRCTAN-COOH 25 38 .times. more Ac-V4(5fW)/H9A
Ac-ICV(5fW)QDWGAHRCT- CONH.sub.2 26 31 .times. more
Ac-V4(5-MeW)/H9A Ac-ICV(5-methyl-W)QDWGAHRCT-CONH.sub.2 27 67
.times. more Ac-V4(1-MeW)/H9A
Ac-ICV(1-methvl-W)QDWGAHRCT-CONH.sub.2 28 264 .times. more
Ac-V4W/W7(5fW)/H9A Ac-ICVWQD(5fW)GAHRCT-CONH.sub.2 29 121 .times.
more Ac-V4(5fW)/W7(5fW)/H9A Ac-ICV(5fW)QD(5fW)GAH RCT- CONH.sub.2
30 NA Ac-V4(5-MeW)/W7(5fW)H9A Ac-ICV(5-methyl-W)QD(5fW)GAHRCT- 31
NA CONH.sub.2 Ac-V4(1MeW)/W7(5fW)/H9A
Ac-ICV(1-methyl-W)QD(5fW)GAHRCT- 32 264 .times. more CONH.sub.2
+G/V4(6fW)/W7(6fW)H9A + N- H-GICV(6fW)QD(6fW)GAHRCTN-COOH 33 126
.times. more OH Ac-V4(1-formyl-W)/H9A
Ac-ICV(1-formyl-W)QDWGAHRCT-CONH.sub.2 34 264 .times. more
Ac-V4(5-methoxy-W)/H9A Ac-ICV(1-methyoxy-W)QDWGAHRCT- 35 76 .times.
more CONH.sub.2 GN4(5f-W)/W7(5fW)/H9A + N-
H-GICV(5fW)QD(5fW)GAHRCTN-COOH 36 112 .times. more OH NA = not
available
[0197] In certain embodiments of the compositions and methods of
the invention the compstatin analog has a sequence selected from
sequences 9-36. In certain embodiments of the compositions and
methods of the invention the compstatin analog has a sequence
selected from SEQ ID NOs: 14, 21, 28, 29, 32, 33, 34, and 36. In
certain embodiments of the compositions and/or methods of the
invention the compstatin analog has a sequence selected from SEQ ID
NOs: 30 and 31. In one embodiment of the compositions and methods
of the invention the compstatin analog has a sequence of SEQ ID NO:
28. In one embodiment of the compositions and methods of the
invention the compstatin analog has a sequence of SEQ ID NO: 32. In
one embodiment of the compositions and methods of the invention the
compstatin analog has a sequence of SEQ ID NO: 34. In one
embodiment of the compositions and methods of the invention the
compstatin analog has a sequence of SEQ ID NO: 36.
[0198] In some embodiments a blocking moiety B.sup.1 comprises an
amino acid, which may be represented as Xaa0. In some embodiments
blocking moiety B.sup.2 comprises an amino acid, which may be
represented as XaaN. In some embodiments blocking moiety B.sup.1
and/or B.sup.2 comprises a non-standard amino acid, such as a
D-amino acid, N-alkyl amino acid (e.g., N-methyl amino acid). In
some embodiments a blocking moiety B.sup.1 and/or B.sup.2 comprises
a non-standard amino acid that is an analog of a standard amino
acid. In some embodiments an amino acid nalog comprises a lower
alkyl, lower alkoxy, or halogen substituent, as compared with a
standard amino acid of which it is an analog. In some embodiments a
substituent is on a side chain. In some embodiments a substituent
is on an alpha carbon atom. In some embodiments, a blocking moiety
B.sup.1 comprising an amino acid, e.g., a non-standard amino acid,
further comprises a moiety B.sup.1a. For example, blocking moiety
B.sup.1 may be represented as B.sup.1a-Xaa0. In some embodiments
B.sup.1a neutralizes or reduces a positive charge that may
otherwise be present at the N-terminus at physiological pH. In some
embodiments B.sup.1a comprises or consists of, e.g., an acyl group
that, e.g., comprises between 1 and 12 carbons, e.g., between 1 and
6 carbons. In certain embodiments blocking moiety B.sup.1a is
selected from the group consisting of: formyl, acetyl, proprionyl,
butyryl, isobutyryl, valeryl, isovaleryl, etc. In some embodiments,
a blocking moiety B.sup.2 comprising an amino acid, e.g., a
non-standard amino acid, may further comprise a moiety B.sup.2a For
example, blocking moiety B.sup.2 may be represented as
XaaN-B.sup.2a, where N represents the appropriate number for the
amino acid (which will depend on the numbering used in the rest of
the peptide). In some embodiments B.sup.2a neutralizes or reduces a
negative charge that may otherwise be present at the C-terminus at
physiological pH. In some embodiments B.sup.2a comprises or
consists of a primary or secondary amine (e.g., NH.sub.2). It will
be understood that a blocking activity of moiety B.sup.1a-Xaa0
and/or XaaN-B.sup.2a may be provided by either or both components
of the moiety in various embodiments. In some embodiments a
blocking moiety or portion thereof, e.g., an amino acid residue,
may contribute to increasing affinity of the compound for C3 or C3b
and/or improve the activity of the compound. In some embodiments a
contribution to affinity or activity of an amino acid residue may
be at least as important as a contribution to blocking activity.
For example, in some embodiments Xaa0 and/or XaaN in B.sup.1a-Xaa0
and/or XaaN-B.sup.2a may function mainly to increase affinity or
activity of the compound, while B.sup.1a and/or B.sup.2a may
inhibit digestion of and/or neutralize a charge of the peptide. In
some embodiments a compstatin analog comprises the amino acid
sequence of any of SEQ ID NOs: 5-36, wherein SEQ ID NOs: 5-36 is
further extended at the N- and/or C-terminus. In some embodiments,
the sequence may be represented as
B.sup.1a-Xaa0-SEQUENCE-XaaN-B.sup.2a, where SEQUENCE represents any
of SEQ ID NOs: 5-36, wherein B.sup.1a and B.sup.2a may
independently be present or absent. For example, in some
embodiments a compstatin analog comprises
B.sup.1a-Xaa0-X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X''aa1-X''aa2-X''aa-
3-X''aa4-X''aa5-XaaN-B.sup.2a (SEQ ID NO: 37), where
X'aa1-X'aa2-X'aa3-X'aa4, Xaa, X''aa1, X''aa2, X''aa3, X''aa4, and
X''aa5 are as set forth above for SEQ ID NO: 5.
[0199] In some embodiments a compstatin analog comprises
B.sup.1a-Xaa0-Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4-X-
aaN-B.sup.2a (SEQ ID NO: 38), where Xaa1, Xaa2, Xaa2*, Xaa3, and
Xaa4 are as set forth above for SEQ ID NO: 6 or wherein Xaa1, Xaa2,
Xaa2*, Xaa3, and Xaa4 are as set forth for SEQ ID NO: 6 or SEQ ID
NO: 7.
[0200] In some embodiments a compstatin analog comprises
B.sup.1a-Xaa0-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xa-
a12-Xaa13-XaaN-B.sup.2a (SEQ ID NO: 39) wherein Xaa1, Xaa2, Xaa3,
Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, and Xaa13
are identical to amino acids at positions 1-13 of any of SEQ ID
NOs: 9-36.
[0201] In some embodiments Xaa0 and/or XaaN in any compstatin
analog sequence comprises an amino acid that comprises an aromatic
ring having an alkyl substituent at one or more positions. In some
embodiments an alkyl substituent is a lower alkyl substituent. For
example, in some embodiments an alkyl substituent is a methyl or
ethyl group. In some embodiments a substituent is located at any
position that does not destroy the aromatic character of the
compound. In some embodiments a substituent is located at any
position that does not destroy the aromatic character of a ring to
which the substituent is attached. In some embodiments a
substituent is located at position 1, 2, 3, 4, or 5. In some
embodiments Xaa0 comprises an O-methyl analog of tyrosine,
2-hydroxyphenylalanine or 3-hydroxyphenylalanine. For purposes of
the present disclosure, a lower case "m" followed by a three letter
amino acid abbreviation may be used to specifically indicate that
the amino acid is an N-methyl amino acid. For example, where the
abbreviation "mGly" appears herein, it denotes N-methyl glycine
(also sometimes referred to as sarcosine or Sar). In some
embodiments Xaa0 is or comprises mGly, Tyr, Phe, Arg, Trp, Thr,
Tyr(Me), Cha, mPhe, mVal, mIle, mAla, DTyr, DPhe, DArg, DTrp, DThr,
DTyr(Me), mPhe, mVal, mIle, DAla, or DCha. For example, in some
embodiments a compstatin analog comprises a peptide having a
sequence
B.sup.1-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B.sup-
.2 (SEQ ID NO: 40) or
B.sup.1-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-mGly-Ala-His-Arg-Cys]-mIle-B.sup-
.2 (SEQ ID NO: 41). The two Cys residues are joined by a disulfide
bond in the active compounds. In some embodiments the peptide is
acetylated at the N-terminus and/or amidated at the C-terminus. In
some embodiments B.sup.1 comprises B.sup.1a-Xaa0 and/or B.sup.2
comprises XaaN-B.sup.2a, as described above. For example, in some
embodiments B.sup.1 comprises or consists of Gly, mGly, Tyr, Phe,
Arg, Trp, Thr, Tyr(Me), mPhe, mVal, mIle, mAla, DTyr, DPhe, DTrp,
DCha, DAla and B.sup.2 comprises NH.sub.2, e.g., a carboxy terminal
--OH of mIle is replaced by NH.sub.2. In some embodiments B.sup.1
comprises or consists of mGly, Tyr, DTyr, or Tyr(Me) and B.sup.2
comprises NH.sub.2, e.g., a carboxy terminal --OH of mIle is
replaced by NH.sub.2. In some embodiments an Ile at position Xaa1
is replaced by Gly. Complement inhibition potency and/or C3b
binding parameters of selected compstatin analogs are described in
WO/2010/127336 (PCT/US2010/033345) and/or in Qu, et al.,
Immunobiology (2012), doi:10.1016/j.imbio.2012.06.003.
[0202] In some embodiments a blocking moiety or portion thereof,
e.g., an amino acid residue, may contribute to increasing affinity
of the compound for C3 or C3b and/or improve the activity of the
compound. In some embodiments a contribution to affinity or
activity of an amino acid or amino acid analog may be more
significant than a blocking activity.
[0203] In certain embodiments of the compositions and methods of
the invention the compstatin analog has a sequence as set forth in
Table 2, but where the Ac-group is replaced by an alternate
blocking moiety B.sup.1, as described herein. In some embodiments
the --NH.sub.2 group is replaced by an alternate blocking moiety
B.sup.2, as described herein.
[0204] In one embodiment, the compstatin analog binds to
substantially the same region of the .beta. chain of human C3 as
does compstatin. In one embodiment the compstatin analog is a
compound that binds to a fragment of the C-terminal portion of the
.beta. chain of human C3 having a molecular weight of about 40 kDa
to which compstatin binds (Soulika, A. M., et al., Mol. Immunol.,
35:160, 1998; Soulika, A. M., et al., Mol. Immunol. 43(12):2023-9,
2006). In certain embodiments the compstatin analog is a compound
that binds to the binding site of compstatin as determined in a
compstatin-C3 structure, e.g., a crystal structure or NMR-derived
3D structure. In certain embodiments the compstatin analog is a
compound that could substitute for compstatin in a compstatin-C3
structure and would form substantially the same intermolecular
contacts with C3 as compstatin. In certain embodiments the
compstatin analog is a compound that binds to the binding site of a
peptide having a sequence set forth in Table 2, e.g., SEQ ID NO:
14, 21, 28, 29, 32, 33, 34, or 36 or another compstatin analog
sequence disclosed herein in a peptide-C3 structure, e.g., a
crystal structure. In certain embodiments the compstatin analog is
a compound that binds to the binding site of a peptide having SEQ
ID NO: 30 or 31 in a peptide-C3 structure, e.g., a crystal
structure. In certain embodiments the compstatin analog is a
compound that could substitute for the peptide of SEQ ID NO: 9-36,
e.g., a compound that could substitute for the peptide of SEQ ID
NO: 14, 21, 28, 29, 32, 33, 34, or 36 or another compstatin analog
sequence disclosed herein in a peptide-C3 structure and would form
substantially the same intermolecular contacts with C3 as the
peptide. In certain embodiments the compstatin analog is a compound
that could substitute for the peptide of SEQ ID NO: 30 or 31 in a
peptide-C3 structure and would form substantially the same
intermolecular contacts with C3 as the peptide.
[0205] One of ordinary skill in the art will readily be able to
determine whether a compstatin analog binds to a fragment of the
C-terminal portion of the .beta. chain of C3 using routine
experimental methods. For example, one of skill in the art could
synthesize a photocrosslinkable version of the compstatin analog by
including a photo-crosslinking amino acid such as
p-benzoyl-L-phenylalanine (Bpa) in the compound, e.g., at the
C-terminus of the sequence (Soulika, A. M., et al, supra).
Optionally additional amino acids, e.g., an epitope tag such as a
FLAG tag or an HA tag could be included to facilitate detection of
the compound, e.g., by Western blotting. The compstatin analog is
incubated with the fragment and crosslinking is initiated.
Colocalization of the compstatin analog and the C3 fragment
indicates binding. Surface plasmon resonance may also be used to
determine whether a compstatin analog binds to the compstatin
binding site on C3 or a fragment thereof. One of skill in the art
would be able to use molecular modeling software programs to
predict whether a compound would form substantially the same
intermolecular contacts with C3 as would compstatin or a peptide
having the sequence of any of the peptides in Table 2, e.g., SEQ ID
NO: 14, 21, 28, 29, 32, 33, 34, or 36, or in some embodiments SEQ
ID NO: 30 or 31 or another compstatin analog sequence disclosed
herein.
[0206] Compstatin analogs may be prepared by various synthetic
methods of peptide synthesis known in the art via condensation of
amino acid residues, e.g., in accordance with conventional peptide
synthesis methods, may be prepared by expression in vitro or in
living cells from appropriate nucleic acid sequences encoding them
using methods known in the art. For example, peptides may be
synthesized using standard solid-phase methodologies as described
in Malik, supra, Katragadda, supra, WO2004026328, and/or
WO2007062249. Potentially reactive moieties such as amino and
carboxyl groups, reactive functional groups, etc., may be protected
and subsequently deprotected using various protecting groups and
methodologies known in the art. See, e.g., "Protective Groups in
Organic Synthesis", 3.sup.rd ed. Greene, T. W. and Wuts, P. G.,
Eds., John Wiley & Sons, New York: 1999. Peptides may be
purified using standard approaches such as reversed-phase HPLC.
Separation of diasteriomeric peptides, if desired, may be performed
using known methods such as reversed-phase HPLC. Preparations may
be lyophilized, if desired, and subsequently dissolved in a
suitable solvent, e.g., water. The pH of the resulting solution may
be adjusted, e.g. to physiological pH, using a base such as NaOH.
Peptide preparations may be characterized by mass spectrometry if
desired, e.g., to confirm mass and/or disulfide bond formation.
See, e.g., Mallik, 2005, and Katragadda, 2006.
[0207] A compstatin analog can be modified by addition of a
molecule such as polyethylene glycol (PEG) or similar molecules to
stabilize the compound, reduce its immunogenicity, increase its
lifetime in the body, increase or decrease its solubility, and/or
increase its resistance to degradation. Methods for pegylation are
well known in the art (Veronese, F. M. & Harris, Adv. Drug
Deliv. Rev. 54, 453-456, 2002; Davis, F. F., Adv. Drug Deliv. Rev.
54, 457-458, 2002); Hinds, K. D. & Kim, S. W. Adv. Drug Deliv.
Rev. 54, 505-530 (2002; Roberts, M. J., Bentley, M. D. &
Harris, J. M. Adv. Drug Deliv. Rev. 54, 459-476; 2002); Wang, Y. S.
et al. Adv. Drug Deliv. Rev. 54, 547-570, 2002). A wide variety of
polymers such as PEGs and modified PEGs, including derivatized PEGs
to which polypeptides can conveniently be attached are described in
Nektar Advanced Pegylation 2005-2006 Product Catalog, Nektar
Therapeutics, San Carlos, Calif., which also provides details of
appropriate conjugation procedures. In another embodiment a
compstatin analog is fused to the Fc domain of an immunoglobulin or
a portion thereof. In some other embodiments a compstatin analog is
conjugated to an albumin moiety or to an albumin binding peptide.
Thus in some embodiments a compstatin analog is modified with one
or more polypeptide or non-polypeptide components, e.g., the
compstatin analog is pegylated or conjugated to another moiety. In
some embodiments the component is not the Fc domain of an
immunoglobulin or a portion thereof. A compstatin analog can be
provided as a multimer or as part of a supramolecular complex,
which can include either a single molecular species or multiple
different species (e.g., multiple different analogs).
[0208] In some embodiments, a compstatin analog of use in methods
described herein is a long-acting compstatin analog, that has a
terminal half-life of at least 3, 4, 5, 6, or 7 days. In some
embodiments a long-acting compstatin analog is a pegylated
compstatin analog. Exemplary long-acting compstatin analogs are
described below and/or in PCT/US12/37648, entitled "CELL-REACTIVE,
LONG-ACTING, OR TARGETED COMPSTATIN ANALOGS AND USES THEREOF",
filed May 11, 2012. In some embodiments of any method or
composition relating to a compstatin analog, the compstatin analog
comprises a compstatin analog whose sequence comprises any of SEQ
ID NOs: 3-41, wherein the compstatin analog is a long-acting
compstatin analog.
[0209] In some embodiments, a compstatin analog is a multivalent
compound comprising a plurality of compstatin analog moieties
covalently or noncovalently linked to a polymeric backbone or
scaffold. The compstatin analog moieties can be identical or
different. In certain embodiments of the invention the multivalent
compound comprises multiple instances, or copies, of a single
compstatin analog moiety. In other embodiments of the invention the
multivalent compound comprises one or more instances of each of two
of more non-identical compstatin analog moieties, e.g., 3, 4, 5, or
more different compstatin analog moieties. In certain embodiments
of the invention the number of compstatin analog moieties ("n") is
between 2 and 6. In other embodiments of the invention n is between
7 and 20. In other embodiments of the invention n is between 20 and
100. In other embodiments n is between 100 and 1,000. In other
embodiments of the invention n is between 1,000 and 10,000. In
other embodiments n is between 10,000 and 50,000. In other
embodiments n is between 50,000 and 100,000. In other embodiments n
is between 100,000 and 1,000,000.
[0210] The compstatin analog moieties may be attached directly to
the polymeric scaffold or may be attached via a linking moiety that
connects the compstatin analog moiety to the polymeric scaffold.
The linking moiety may be attached to a single compstatin analog
moiety and to the polymeric scaffold. Alternately, a linking moiety
may have multiple compstatin analog moieties joined thereto so that
the linking moiety attaches multiple compstatin analog moieties to
the polymeric scaffold.
[0211] In some embodiments, a compstatin analog comprises an amino
acid having a side chain comprising a primary or secondary amine,
e.g., a Lys residue. For example, any of the compstatin analog
sequences disclosed herein may be extended or modified by addition
of a linker comprising one or more amino acids, e.g., one or more
amino acids comprising a primary or secondary amine, e.g., in a
side chain thereof. For example, a Lys residue, or a sequence
comprising a Lys residue, is added at the N-terminus and/or
C-terminus of the compstatin analog. In some embodiments, the Lys
residue is separated from the cyclic portion of the compstatin
analog by a rigid or flexible spacer. A linker or spacer may, for
example, comprise a substituted or unsubstituted, saturated or
unsaturated alkyl chain, oligo(ethylene glycol) chain, and/or other
moieties. The length of the chain may be, e.g., between 2 and 20
carbon atoms. In some embodiments the spacer is or comprises a
peptide. The peptide spacer may be, e.g., between 1 and 20 amino
acids in length, e.g., between 4 and 20 amino acids in length.
Suitable spacers can comprise or consist of multiple Gly residues,
Ser residues, or both, for example. Optionally, the amino acid
having a side chain comprising a primary or secondary amine and/or
at least one amino acid in a spacer is a D-amino acid. A PEG moiety
or similar molecule or polymeric scaffold may be linked to the
primary or secondary amine, optionally via a linker. In some
embodiments, a bifunctional linker is used. A bifunctional linker
may comprise two reactive functional groups, which may be the same
or different in various embodiments. In various embodiments, one or
more linkers, spacers, and/or techniques of conjugation described
in Hermanson, supra, is used.
[0212] Any of a variety of polymeric backbones or scaffolds could
be used. For example, the polymeric backbone or scaffold may be a
polyamide, polysaccharide, polyanhydride, polyacrylamide,
polymethacrylate, polypeptide, polyethylene oxide, or dendrimer.
Suitable methods and polymeric backbones are described, e.g., in
WO98/46270 (PCT/US98/07171) or WO98/47002 (PCT/US98/06963). In one
embodiment, the polymeric backbone or scaffold comprises multiple
reactive functional groups, such as carboxylic acids, anhydride, or
succinimide groups. The polymeric backbone or scaffold is reacted
with the compstatin analogs. In one embodiment, the compstatin
analog comprises any of a number of different reactive functional
groups, such as carboxylic acids, anhydride, or succinimide groups,
which are reacted with appropriate groups on the polymeric
backbone. Alternately, monomeric units that could be joined to one
another to form a polymeric backbone or scaffold are first reacted
with the compstatin analogs and the resulting monomers are
polymerized. In another embodiment, short chains are
prepolymerized, functionalized, and then a mixture of short chains
of different composition are assembled into longer polymers.
[0213] In some aspects a moiety such as a polyethylene glycol (PEG)
chain or other polymer(s) that, e.g., stabilize the compound,
increase its lifetime in the body, increase its solubility,
decrease its immunogenicity, and/or increase its resistance to
degradation may be referred to herein as a "clearance reducing
moiety" (CRM), and a compstatin analog comprising such a moiety may
be referred to as a long-acting compstatin analog.
[0214] In some aspects, a long-acting compstatin analog comprises a
compound of formula M-L-A, wherein A is a moiety that comprises a
CRM, L is an optionally present linking portion, and M comprises a
compstatin analog moiety. The compstatin analog moiety can comprise
any compstatin analog, e.g., any compstatin analog described above,
in various embodiments. Formula M-L-A encompasses embodiments in
which L-A is present at the N-terminus of the compstatin analog
moiety, embodiments in which L-A is present at the C-terminus of
the compstatin analog moiety, embodiments in which L-A is attached
to a side chain of an amino acid of the compstatin analog moiety,
and embodiments where the same or different L-As are present at
both ends of M. It will be appreciated that when certain compstatin
analog(s) are present as a compstatin analog moiety in a compound
of formula M-L-A, a functional group of the compstatin analog will
have reacted with a functional group of L to form a covalent bond
to A or L. For example, a long-acting compstatin analog in which
the compstatin analog moiety comprises a compstatin analog that
contains an amino acid with a side chain containing a primary amine
(NH.sub.2) group (which compstatin analog can be represented by
formula R.sup.1--(NH.sub.2)), can have a formula R.sup.1--NH-L-A in
which a new covalent bond to L (e.g., N--C) has been formed and a
hydrogen lost. Thus the term "compstatin analog moiety" includes
molecular structures in which at least one atom of a compstatin
analog participates in a covalent bond with a second moiety, which
may, e.g., modification of a side chain. Similar considerations
apply to compstatin analog moieties present in multivalent
compounds. In some embodiments, a blocking moiety at the N-terminus
or C-terminus of a compstatin analog is replaced by L-A in the
structure of a long-acting compstatin analog.
[0215] In some embodiments, L comprises an unsaturated moiety such
as --CH.dbd.CH-- or --CH.sub.2--CH.dbd.CH--; a moiety comprising a
non-aromatic cyclic ring system (e.g., a cyclohexyl moiety), an
aromatic moiety (e.g., an aromatic cyclic ring system such as a
phenyl moiety); an ether moiety (--C--O--C--); an amide moiety
(--C(.dbd.O)--N--); an ester moiety (--CO--O--); a carbonyl moiety
(--C(.dbd.O)--); an imine moiety (--C.dbd.N--); a thioether moiety
(--C--S--C--); an amino acid residue; and/or any moiety that can be
formed by the reaction of two compatible reactive functional
groups. In certain embodiments, one or more moieties of a linking
portion is/are substituted by independent replacement of one or
more of the hydrogen (or other) atoms thereon with one or more
moieties including, but not limited to aliphatic; aromatic, aryl;
alkyl, aralkyl, alkanoyl, aroyl, alkoxy; thio; F; Cl; Br; I; --NO2;
--CN; --CF3; --CH2CF3; --CHCl2; --CH2OH; --CH2CH2OH; --CH2NH2;
--CH2SO2CH3; - or -GRG1 wherein G is --O--, --S--, --NRG2-,
--C(.dbd.O)--, --S(.dbd.O)--, --SO2-, --C(.dbd.O)O--,
--C(.dbd.O)NRG2-, --OC(.dbd.O)--, --NRG2C(.dbd.O)--,
--OC(.dbd.O)O--, --OC(.dbd.O)NRG2-, --NRG2C(.dbd.O)O--,
--NRG2C(.dbd.O)NRG2-, --C(.dbd.S)--, --C(.dbd.S)S--,
--SC(.dbd.S)--, --SC(.dbd.S)S--, --C(.dbd.NRG2)-,
--C(.dbd.NRG2)O--, --C(.dbd.NRG2)NRG3-, --OC(.dbd.NRG2)-,
--NRG2C(.dbd.NRG3)-, --NRG2SO2-, --NRG2SO2NRG3-, or --SO2NRG2-,
wherein each occurrence of RG1, RG2 and RG3 independently includes,
but is not limited to, hydrogen, halogen, or an optionally
substituted aliphatic, aromatic, or aryl moiety. It will be
appreciated that cyclic ring systems when present as substituents
may optionally be attached via a linear moiety. Combinations of
substituents and variables envisioned by this invention are
preferably those that result in the formation of stable compounds
useful in any one or more of the methods described herein, e.g.,
useful for the treatment of one or more disorders and/or for
contacting a cell, tissue, or organ, as described herein, and/or
useful as intermediates in the manufacture of one or more such
compounds.
[0216] L can comprise one or more of any of the moieties described
in the preceding paragraph, in various embodiments. In some
embodiments, L comprises two or more different moieties linked to
one another to form a structure typically having a length of
between 1 to about 60 atoms, between 1 to about 50 atoms, e.g.,
between 1 and 40, between 1 and 30, between 1 and 20, between 1 and
10, or between 1 and 6 atoms, where length refers to the number of
atoms in the main (longest) chain. In some embodiments, L comprises
two or more different moieties linked to one another to form a
structure typically having between 1 to about 40, e.g., between 1
and 30, e.g., between 1 and 20, between 1 and 10, or between 1 and
6 carbon atoms in the main (longest) chain.
[0217] In some embodiments, a long-acting compstatin analog has an
average plasma half-life of at least 1 day, e.g., 1-3 days, 3-7
days, 7-14 days, or 14-28 days, when administered IV at a dose of
10 mg/kg to humans or to non-human primates. In some embodiments,
average plasma half-life of a long-acting compstatin analog
following administration IV at a dose of 10 mg/kg to humans or to
non-human primates is increased by at least a factor of 2, e.g., by
a factor of 2-5, 5-10, 10-50, or 50-100-fold as compared with that
of a corresponding compstatin analog having the same amino acid
sequence (and, if applicable, one or more blocking moiet(ies)) but
not comprising the CRM.
[0218] In some embodiments, a plasma half-life is a terminal
half-life after administration of a single IV dose. In some
embodiments, a plasma half-life is a terminal half-life after
steady state has been reached following administration of multiple
IV doses. In some embodiments, a long-acting compstatin analog
achieves a Cmax in plasma at least 5-fold as great as that of a
corresponding compstatin analog not comprising the CRM, e.g.,
between 5- and 50-fold as great, following administration of a
single IV dose to a primate, or following administration of
multiple IV doses. In some embodiments, a long-acting compstatin
analog achieves a Cmax in plasma between 10- and 20-fold as great
as that of a corresponding compstatin analog not comprising the CRM
following administration of a single IV dose to a primate, or
following administration of multiple IV doses. In some embodiments
a primate is human. In some embodiments a primate is a non-human
primate, e.g., a monkey, such as a Cynomolgus monkey or Rhesus
monkey. In some embodiments, renal clearance of a long-acting
compstatin analog during the first 24 hours following
administration IV at a dose of 10 mg/kg to humans or to non-human
primates is reduced by at least a factor of 2, e.g., by a factor of
2-5, 5-10, 10-50, or 50-100-fold as compared with renal clearance
of a corresponding compstatin analog. The concentration of
compstatin analog can be measured in blood and/or urine samples
using, e.g., UV, HPLC, mass spectrometry (MS) or antibody to the
CRM, or combinations of such methods, such as LC/MS or LC/MS/MS.
Pharmacokinetic parameters such as half-life and clearance can be
determined using methods known to those of ordinary skill in the
art. Pharmacokinetic analysis can be performed, e.g., with
WinNonlin software v 5.2 (Pharsight Corporation, St. Louis,
Mo.).
[0219] In some embodiments, a long-acting compstatin analog has a
molar activity of at least about 10%, 20%, 30%, e.g., between 30%
and 40%, between 30% and 50%, between 30% and 60%, between 30% and
70%, between 30% and 80%, between 30% and 90%, or more, of the
activity of a corresponding compstatin analog having the same amino
acid sequence (and, if applicable, one or more blocking moiet(ies))
but not comprising a CRM. In some embodiments wherein a long-acting
compstatin analog comprises multiple compstatin analog moieties,
the molar activity of the long-acting compstatin analog is at least
about 10%, 20%, or 30%, e.g., between 30% and 40%, between 30% and
50%, between 30% and 60%, between 30% and 70%, between 30% and 80%,
between 30% and 90%, or more, of the sum of the activities of said
compstatin analog moieties. In some embodiments, a polyethylene
glycol (PEG) comprises a (CH.sub.2CH.sub.2O).sub.m moiety having a
molecular weight of at least 500 daltons. In some embodiments, a
linker comprises an (CH.sub.2CH.sub.2O).sub.n moiety having an
average molecular weight of between about 500; 1,000; 1,500; 2,000;
5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000;
80,000; 90,000; and 100,000 daltons. "Average molecular weight"
refers to the number average molecular weight. In some embodiments,
the polydispersity D of a (CH.sub.2CH.sub.2O)n moiety is between
1.0005 and 1.50, e.g., between 1.005 and 1.10, 1.15, 1.20, 1.25,
1.30, 1.40, or 1.50, or any value between 1.0005 and 1.50.
[0220] In some embodiments, a (CH.sub.2CH.sub.2O)n moiety is
monodisperse and the polydispersity of a (CH.sub.2CH.sub.2O)n
moiety is 1.0. Such monodisperse (CH.sub.2CH.sub.2O)n moieties are
known in the art and are commercially available from Quanta
BioDesign (Powell, Ohio), and include, by way of nonlimiting
example, monodisperse moieties where n is 2, 4, 6, 8, 12, 16, 20,
or 24.
[0221] In some embodiments, a compound comprises multiple
(CH.sub.2CH.sub.2O).sub.n moieties wherein the total molecular
weight of said (CH.sub.2CH.sub.2O).sub.n moieties is between about
1,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000;
70,000; 80,000; 90,000; and 100,000 daltons. In some embodiments,
the compound comprises multiple (CH.sub.2CH.sub.2O).sub.n moieties
having defined lengths, e.g., n=4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, or 30 or more. In some embodiments, the compound
comprises a sufficient number of (CH.sub.2CH.sub.2O).sub.n moieties
having defined lengths to result in a total molecular weight of
said (CH.sub.2CH.sub.2O).sub.n moieties of between about 1,000;
5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000;
80,000; 90,000; and 100,000 daltons. In some embodiments n is
between about 30 and about 3000. In some embodiments a compstatin
analog moiety is attached at each end of a linear PEG. A
bifunctional PEG having a reactive functional group at each end of
the chain may be used, e.g., as described above. In some
embodiments the reactive functional groups are identical while in
some embodiments different reactive functional groups are present
at each end. In some embodiments, multiple
(CH.sub.2CH.sub.2O).sub.n moieties are provided as a branched
structure. The branches may be attached to a linear polymer
backbone (e.g., as a comb-shaped structure) or may emanate from one
or more central core groups, e.g., as a star structure. In some
embodiments, a branched molecule has 3 to 10
(CH.sub.2CH.sub.2O).sub.n chains. In some embodiments, a branched
molecule has 4 to 8 (CH.sub.2CH.sub.2O).sub.n chains. In some
embodiments, a branched molecule has 10, 9, 8, 7, 6, 5, 4, or 3
(CH.sub.2CH.sub.2O).sub.n chains. In some embodiments, a
star-shaped molecule has 10-100, 10-50, 10-30, or 10-20
(CH.sub.2CH.sub.2O).sub.n chains emanating from a central core
group. In some embodiments a long-acting compstatin analog thus may
comprise, e.g., 3-10 compstatin analog moieties, e.g., 4-8
compstatin analog moieties, each attached to a
(CH.sub.2CH.sub.2O).sub.n chain via a functional group at the end
of the chain. In some embodiments a long-acting compstatin analog
may comprise, e.g., 10-100 compstatin analog moieties, each
attached to a (CH.sub.2CH.sub.2O).sub.n chain via a functional
group at the end of the chain. In some embodiments, branches
(sometimes referred to as "arms") of a branched or star-shaped PEG
contain about the same number of (CH.sub.2CH.sub.2O) moieties. In
some embodiments, at least some of the branch lengths may differ.
It will be understood that in some embodiments one or more
(CH.sub.2CH.sub.2O).sub.n chains does not have a comptatin analog
moiety attached thereto. In some embodiments at least about 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the chains has a
compstatin analog moiety attached thereto.
[0222] In genera and compounds depicted herein, a polyethylene
glycol moiety is drawn with the oxygen atom on the right side of
the repeating unit or the left side of the repeating unit. In cases
where only one orientation is drawn, the present invention
encompasses both orientations (i.e., (CH.sub.2CH.sub.2O).sub.n and
(OCH.sub.2CH.sub.2).sub.n) of polyethylene glycol moieties for a
given compound or genus, or in cases where a compound or genus
contains multiple polyethylene glycol moieties, all combinations of
orientations are encompasses by the present disclosure.
[0223] Formulas of some exemplary monofunctional PEGs comprising a
reactive functional group are illustrated below. For illustrative
purposes, formulas in which the reactive functional group(s)
comprise an NHS ester are depicted, but other reactive functional
groups could be used, e.g., as described above. In some
embodiments, the (CH.sub.2CH.sub.2O).sub.n are depicted as
terminating at the left end with a methoxy group (OCH.sub.3) but it
will be understood that the chains depicted below and elsewhere
herein may terminate with a different OR moiety (e.g., an aliphatic
group, an alkyl group, a lower alkyl group, or any other suitable
PEG end group) or an OH group. It will also be appreciated that
moieties other than those depicted may connect the
(CH.sub.2CH.sub.2O).sub.n moieties with the NHS group in various
embodiments.
[0224] In some embodiments, a monofunctional PEG is of formula
A:
##STR00001##
wherein "Reactive functional group" and n are as defined above and
described in classes and subclasses herein; [0225] R.sup.1 is
hydrogen, aliphatic, or any suitable end group; and [0226] T is a
covalent bond or a C.sub.1-12 straight or branched, hydrocarbon
chain wherein one or more carbon units of T are optionally and
independently replaced by --O--, --S--, --N(R.sup.x)--, --C(O)--,
--C(O)O--, --OC(O)--, --N(R.sup.x)C(O)--, --C(O)N(R.sup.x)--,
--S(O)--, --S(O).sub.2--, --N(R.sup.x)SO.sub.2--, or
--SO.sub.2N(R.sup.x)--; and [0227] each R.sup.x is independently
hydrogen or C.sub.1-6 aliphatic.
[0228] Exemplary monofunctional PEGs of formula A include:
##STR00002##
[0229] In Formula I, the moiety comprising the reactive functional
group has the general structure --CO--(CH.sub.2).sub.m--COO--NHS,
where m=2. In some embodiments, a monofunctional PEGs has the
structure of Formula I, where m is between 1 and 10, e.g., between
1 and 5. For example, in some embodiments m is 3, as shown
below:
##STR00003##
[0230] In Formula II, the moiety comprising the reactive functional
group has the general structure --(CH.sub.2).sub.m--COO--NHS, where
m=1. In some embodiments a monofunctional PEG has the structure of
Formula II, where m is between 1 and 10 (e.g., wherein m is 5 as
shown in Formula III below), or wherein m is 0 (as shown below in
Formula IIIa).
##STR00004##
[0231] In some embodiments a bifunctional linear PEG comprises a
moiety comprising a reactive functional group at each of its ends.
The reactive functional groups may be the same (homobifunctional)
or different (heterobifunctional). In some embodiments the
structure of a bifunctional PEG may be symmetric, wherein the same
moiety is used to connect the reactive functional group to oxygen
atoms at each end of the --(CH.sub.2CH.sub.2O).sub.n chain. In some
embodiments different moieties are used to connect the two reactive
functional groups to the PEG portion of the molecule. The
structures of exemplary bifunctional PEGs are depicted below. For
illustrative purposes, formulas in which the reactive functional
group(s) comprise an NHS ester are depicted, but other reactive
functional groups could be used.
[0232] In some embodiments, a bifunctional linear PEG is of formula
B:
##STR00005##
wherein each T and "Reactive functional group" is independently as
defined above and described in classes and subclasses herein, and n
is as defined above and described in classes and subclasses
herein.
[0233] Exemplary bifunctional PEGs of formula B include:
##STR00006##
[0234] In Formula IV, the moiety comprising the reactive functional
group has the general structure --(CH.sub.2).sub.m--COO--NHS, where
m=1. In some embodiments, a bifunctional PEGs has the structure of
Formula IV, where m is between 1 and 10, e.g., between 1 and 5.
##STR00007##
[0235] In Formula V, the moiety comprising the reactive functional
group has the general structure --CO--(CH.sub.2).sub.m--COO--NHS,
where m=2. In some embodiments, a bifunctional PEGs has the
structure of Formula V, where m is between 1 and 10, e.g., between
1 and 5.
[0236] In some embodiments, a branched, comb, or star-shaped PEG
comprises a moiety comprising a reactive functional group at the
end of each of multiple --(CH.sub.2CH.sub.2O).sub.n chains. The
reactive functional groups may be the same or there may be at least
two different groups. In some embodiments, a branched, comb, or
star-shaped PEG is of the following formulae:
##STR00008##
[0237] wherein each R.sup.2 is independently a "Reactive functional
group" or R.sup.1, and each T, n, and "Reactive functional group"
is independently as defined above and described in classes and
subclasses herein. The structure of exemplary branched PEGs (having
8 arms, or branches) comprising NHS moieties as reactive functional
groups is depicted below:
##STR00009##
[0238] The structure of exemplary branched PEGs (having 4 arms, or
branches) comprising NHS moieties as reactive functional groups is
depicted below:
##STR00010##
[0239] The number of branches emanating from the backbone may be
varied. For example, the number 4 in the above formulae VI and VII
may be changed to any other integer between 0 and 10 in various
embodiments. In certain embodiments, one or more branches does not
contain a reactive function group and the branch terminates with a
--CH.sub.2CH.sub.2OH or --CH.sub.2CH.sub.2OR group, as described
above.
[0240] In some embodiments a branched PEG has the structure of
Formula VII, VIII, or IX (or variants thereof having different
numbers of branches) with the proviso that x is
##STR00011##
[0241] In some embodiments a branched PEG has the structure of
Formula VII, VIII, or IX (or variants thereof having different
numbers of branches) with the proviso that x is
##STR00012##
[0242] Of course the methylene (CH.sub.2) group in the above x
moiety may instead comprise a longer alkyl chain (CH2).sub.m, where
m is up to 2, 3, 4, 5, 6, 8, 10, 20, or 30, or may comprise one or
more other moieties described herein.
[0243] In some embodiments, exemplary branched PEGs having NHS or
maleimde reactive groups are depicted below:
##STR00013##
[0244] In some embodiments, a variant of Formula X or XI are used,
wherein 3 or each of the 4 branches comprise a reactive functional
group.
[0245] Still other examples of PEGs may be represented as
follows:
##STR00014##
As noted above, it will be appreciated that, as described herein,
in various embodiments any of a variety of moieties may be
incorporated between the peptide component and
(CH.sub.2CH.sub.2O).sub.n--R moiety of a long-acting compstatin
analog, such as an linear alkyl, ester, amide, aromatic ring (e.g.,
a substituted or unsubstituted phenyl), a substituted or
unsubstituted cycloalkyl structure, or combinations thereof. In
some embodiments such moiet(ies) may render the compound more
susceptible to hydrolysis, which may release the peptide portion of
the compound from the CRM. In some embodiments, such release may
enhance the in vivo tissue penetration and/or activity of the
compound. In some embodiments hydrolysis is general (e.g.,
acid-base) hydrolysis. In some embodiments hydrolysis is
enzyme-catalyzed, e.g., esterase-catalyzed. Of course both types of
hydrolysis may occur. Examples of PEGs comprising one or more such
moieties and an NHS ester as a reactive functional group are as
follows:
##STR00015##
[0246] In some embodiments a branched (multi-arm) PEG or
star-shaped PEG comprises a pentaerythritol core, hexaglycerin
core, or tripentaerythritol core. It will be understood that the
branches may not all emanate from a single point in certain
embodiments.
[0247] Monofunctional, bifunctional, branched, and other PEGs
comprising one or more reactive functional groups may be obtained
from, e.g., NOF America Corp. White Plains, N.Y. or BOC Sciences
45-16 Ramsey Road Shirley, N.Y. 11967, USA, among others.
[0248] In some embodiments a compstatin analog of, e.g., any of SEQ
ID NOs: 3-41 is extended by one or more amino acids at the
N-terminus, C-terminus, or both, wherein at least one of the amino
acids has a side chain that comprises a reactive functional group
such as a primary or secondary amine, a sulfhydryl group, a
carboxyl group (which may be present as a carboxylate group), a
guanidino group, a phenol group, an indole ring, a thioether, or an
imidazole ring, wherein the reactive functional group may be used,
e.g., to attach a CRM or moiety comprising a CRM. In some
embodiments, the amino acid(s) is/are L-amino acids. In some
embodiments, any one or more of the amino acid(s) is a D-amino
acid. If multiple amino acids are added, the amino acids can be
independently selected. In some embodiments, the reactive
functional group (e.g., a primary or secondary amine) is used as a
target for addition of a moiety comprising a CRM Amino acids having
a side chain that comprises a primary or secondary amine include
lysine (Lys) and diaminocarboxylic acids of general structure
NH.sub.2(CH.sub.2).sub.nCH(NH.sub.2)COOH such as
2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid (daba),
and ornithine (orn), wherein n=1 (dapa), 2 (daba), and 3 (orn),
respectively. In some embodiments at least one amino acid is
cysteine, aspartic acid, glutamic acid, arginine, tyrosine,
tryptophan, methionine, or histidine. Cysteine has a side chain
comprising a sulfhydryl group. Aspartic acid and glutamic acid have
a side chain comprising a carboxyl group (ionizable to a
carboxylate group). Arginine has a side chain comprising a
guanidino group. Tyrosine has a side chain comprising a phenol
group (ionizable to a phenolate group). Tryptophan has a side chain
comprising an indole ring include, e.g., tryptophan. Methionine has
a side chain comprising a thioether group include, e.g.,
methionine. Histidine has a side chain comprising an imidazole
ring. A wide variety of non-standard amino acids having side chains
that comprise one or more such reactive functional group(s) are
available, including naturally occurring amino acids and amino
acids not found in nature. See, e.g., Hughes, B. (ed.), Amino
Acids, Peptides and Proteins in Organic Chemistry, Volumes 1-4,
Wiley-VCH (2009-2011); Blaskovich, M., Handbook on Syntheses of
Amino Acids General Routes to Amino Acids, Oxford University Press,
2010. Embodiments in which one or more non-standard amino acid(s)
is/are used to provide a target for addition of a moiety comprising
a CRM are encompassed. Any one or more of the amino acid(s) may be
protected as appropriate during synthesis of the compound. For
example, one or more amino acid(s) may be protected during
reaction(s) involving the target amino acid side chain. In some
embodiments, wherein a sulfhydryl-containing amino acid is used as
a target for addition of a moiety comprising a CRM, the sulfhydryl
is protected while the compound is being cyclized by formation of
an intramolecular disulfide bond between other amino acids such as
cysteines.
[0249] In certain discussion herein, an amino acid having a side
chain containing an amine group is used as an example. Analogous
embodiments are encompassed in which an amino acid having a side
chain containing a different reactive functional group is used. In
some embodiments, an amino acid having a side chain comprising a
primary or secondary amine is attached directly to the N-terminus
or C-terminus of any of SEQ ID NOs: 3-41 via a peptide bond. In
some embodiments, an amino acid having a side chain comprising a
primary or secondary amine is attached to the N- or C-terminus of
any of SEQ ID NOs: 3-41 via a linking portion, which may contain
any one or more of the linking moieties described above. In some
embodiments, at least two amino acids are appended to either or
both termini. The two or more appended amino acids may be joined to
each other by peptide bonds or at least some of the appended amino
acids may be joined to each other by a linking portion, which may
contain any one or more of the linking moieties described
herein.
[0250] It will be understood that a corresponding compstatin analog
not comprising the CRM may also lack one or more such amino acids
which are present in the long-acting compstatin analog to which it
corresponds. Thus, a corresponding compstatin analog comprising any
of SEQ ID NOs: 3-41 and lacking a CRM will be understood to "have
the same amino acid sequence" as SEQ ID NO: 3-41, respectively. For
example, a corresponding compstatin analog comprising the amino
acid sequence of SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 and
lacking a CRM will be understood to "have the same amino acid
sequence" as SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36,
respectively.
[0251] For descriptive purposes a peptide having the amino acid
sequence Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr
(SEQ ID NO: 42) (corresponding to the compstatin analog of SEQ ID
NO: 28, wherein asterisks in SEQ ID NO: 42 represent cysteines
joined by a disulfide bond in the active compound, and (1Me)Trp
represents 1-methyl-tryptophan)), is used as an exemplary
compstatin analog moiety; (CH2).sub.n and (O--CH2-CH2).sub.n are
used as examples of linking portions; lysine is used as an example
of an amino acid comprising a reactive functional group (in some
compounds), and acetylation and amidation of the N- and C-termini,
respectively, are used as optionally present exemplary blocking
moieties in some compounds and may be represented in italics, i.e.,
as Ac and NH.sub.2 respectively. In some embodiments, SEQ ID NO: 42
is extended to comprise a Lys residue at the N- or C-terminus of
the peptide, e.g., as exemplified below for a C-terminal
linkage:
TABLE-US-00003 (SEQ ID NO: 43)
Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-
Arg-Cys*-Thr-Lys-NH.sub.2.
[0252] In some embodiments, a Lys residue is attached to the N- or
C-terminus of SEQ ID NO: 42 via a peptide linker, e.g., as
exemplified below for a C-terminal linkage:
TABLE-US-00004 (SEQ ID NO: 44)
Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-
Arg-Cys*-Thr-(Gly).sub.5-Lys-NH.sub.2.
[0253] In some embodiments, a linker comprising a primary or
secondary amine is added to the N- or C-terminus of a compstatin
analog. In some embodiments, the linker comprises an alkyl chain
and/or an oligo(ethylene glycol) moiety. For example,
NH.sub.2(CH.sub.2CH.sub.2O)nCH.sub.2C(.dbd.O)OH (e.g.,
8-amino-3,6-dioxaoctanoic acid (AEEAc) or
11-amino-3,6,9-trioxaundecanoic acid) or an NHS ester thereof
(e.g., an NHS ester of 8-amino-3,6-dioxaoctanoic acid or
11-amino-3,6,9-trioxaundecanoic acid), can be used. In some
embodiments, the resulting compound is as follows (wherein the
portion contributed by the linker is shown in bold):
TABLE-US-00005 (SEQ ID NO: 45)
NH.sub.2(CH.sub.2).sub.5C(.dbd.O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-
Gly-Ala-His-Arg-Cys-Thr-NH.sub.2. (SEQ ID NO: 46)
NH.sub.2(CH.sub.2CH.sub.2O).sub.2CH.sub.2C(.dbd.O)-Ile-Cys-Val-(1Me)Trp-Gl-
n- Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH.sub.2
[0254] In some embodiments, a Lys residue is attached to the N- or
C-terminus of SEQ ID NO: 42 via a linker comprising a non-peptide
portion. For example, the linker can comprise an alkyl chain,
oligo(ethylene glycol) chain, and/or cyclic ring system. In some
embodiments, 8-AEEAc or an NHS ester thereof is used, resulting (in
the case of attachment of Lys at the C-terminus) in the following
compound (wherein the portion contributed by 8-AEEAc is shown in
bold):
TABLE-US-00006 (SEQ ID NO: 47)
Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-
Arg-Cys*-Thr-NH-CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2-C(.dbd.O)-Lys-N-
H.sub.2
[0255] It will be appreciated that in SEQ ID NOs: 45 and 46, a
--C(.dbd.O) moiety is attached to the adjacent Ile residue via a
C--N bond, wherein the N is part of the amino acid and is not
shown. Similarly, in SEQ ID NO: 47, a --C(.dbd.O) moiety is
attached to the adjacent Lys residue via a C--N bond, wherein the N
is part of the amino acid and is not shown. It will also be
appreciated that that in SEQ ID NO: 47 the NH moiety is attached to
the immediately N-terminal amino acid (Thr), via a C--N bond,
wherein the C is the carbonyl carbon of the amino acid and is not
shown.
[0256] The compounds of SEQ ID NOs: 43-47 can be modified at the
primary amine group to produce a long-acting compstatin analog.
[0257] Exemplary long-acting compstatin analogs are set forth
below, wherein n is sufficient to provide an average molecular
weight of between about 500; 1,000; 1,500; 2,000; 5,000; 10,000;
20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; and
100,000 daltons.
TABLE-US-00007 (SEQ ID NO: 48)
(CH.sub.2CH.sub.2O).sub.nC(.dbd.O)-Ile-Cys-Val-(1Me)Trp-Gln-Asp-Trp-
Gly-Ala-His-Arg-Cys-Thr- NH.sub.2) (SEQ ID NO: 49)
Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His- Arg-Cys*-Thr
-NH-CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2-C(.dbd.O)-Lys-
C(.dbd.O)-(CH.sub.2CH.sub.2O).sub.n-NH.sub.2 (SEQ ID NO: 50)
Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-
Arg-Cys*-Thr-Lys-C(.dbd.O)-(CH.sub.2CH.sub.2O).sub.n -NH.sub.2.
(SEQ ID NO: 51) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-
Arg-Cys*-Thr-(Gly).sub.5-Lys-C(.dbd.O)-(CH.sub.2CH.sub.2O).sub.n-NH.sub.2
(SEQ ID NO: 52)
Ac-(CH.sub.2CH.sub.2O).sub.nC(.dbd.O)Lys-(Gly).sub.5-Ile-
Cys*-Va1-(1Me) Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr - NH.sub.2)
(SEQ ID NO: 53) Ac-(CH.sub.2CH.sub.2O).sub.nC(.dbd.O)Lys-Ile-
Cys*-Val-(1Me)Trp-Gln- Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr -
NH.sub.2)
[0258] In SEQ ID NO: 48, the (CH.sub.2CH.sub.2O)n is coupled via an
amide bond to the N-terminal amino acid. In SEQ ID NOs: 49-53, the
(CH.sub.2CH.sub.2O)n moiety is coupled via an amide bond to a Lys
side chain; thus it will be understood that the NH.sub.2 at the
C-terminus in SEQ ID NOs: 49, 50, and 51, represents amidation of
the C-terminus of the peptide, and it will be understood that in
SEQ ID NOs: 52 and 53, the Ac at the N-terminus represents
acetylation of the N-terminus of the peptide, as described above.
It will also be appreciated by those of ordinary skill in the art
that a free end of a (CH.sub.2CH.sub.2O).sub.n moiety typically
terminates with an (OR) where the underlined O represents the O
atom in the terminal (CH.sub.2CH.sub.2O) group. (OR) is often a
moiety such as a hydroxyl (OH) or methoxy (--OCH.sub.3) group
though other groups (e.g., other alkoxy groups) could be used. Thus
SEQ ID NO: 49, for example, may be represented as
Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-NH--CH.sub.-
2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2--C(.dbd.O)-Lys-(C(.dbd.O)--(CH.sub.2CH-
.sub.2O).sub.n--R)--NH2 (SEQ ID NO: 54) wherein R is, e.g., either
H or CH.sub.3 in the case of a linear PEG. In the case of a
bifunctional, branched or star-shaped PEG, R represents the
remainder of the molecule. Further, it will be understood that the
moiety comprising the reactive functional group may vary, as
described herein (e.g., according to any of the formulas described
herein). For example, long-acting compstatin analogs comprising the
same peptide sequence as SEQ ID NO: 54, in which the moiety
comprising the reactive functional group comprises an ester and/or
alkyl chain may be represented as follows
TABLE-US-00008 (SEQ ID NO: 55)
Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-
Arg-Cys*-Thr-NH-CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2-C(.dbd.O)-Lys-
(C(.dbd.O)-(CH.sub.2).sub.m-(CH.sub.2CH.sub.2O).sub.n-R)-NH.sub.2;
(SEQ ID NO: 56) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-
Arg-Cys*-Thr-NH-CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2-C(.dbd.O)-Lys-
(C(.dbd.O)-(CH.sub.2).sub.mC(.dbd.O)-(CH.sub.2CH.sub.2O).sub.n-R)-NH.sub.2
(SEQ ID NO: 57) Ac-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-
Arg-Cys*-Thr-NH-CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2-C(.dbd.O)-Lys-
(C(.dbd.O)-(CH.sub.2).sub.mC(.dbd.O)-(CH.sub.2)j(CH.sub.2CH.sub.2O).sub.n--
R)-NH.sub.2
[0259] In SEQ ID NOs: 55-57 m may range from 1 up to about 2, 3, 4,
5, 6, 7, 8, 10, 15, 20, or 30 in various embodiments, In SEQ ID
NOs: 57 j may range from 1 up to about 2, 3, 4, 5, 6, 7, 8, 10, 15,
20, or 30 in various embodiments. It will also be appreciated that,
as described herein, in various embodiments other moieties may be
incorporated between the Lys-(C(.dbd.O)-- and
(CH.sub.2CH.sub.2O).sub.n--R, such as an amide, aromatic ring
(e.g., a substituted or unsubstituted phenyl), or a substituted or
unsubstituted cycloalkyl structure.
[0260] In some embodiments a long-acting compstatin analog
comprises a variant of SEQ ID NOs: 48-57 in which
-Ile-Cys*-Val-(1Me)Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-SEQ ID
NO: 72) is replaced by an amino acid sequence comprising the amino
acid sequence of any other compstatin analog, e.g., of any of SEQ
ID NOs 3-27 or 29-41, with the proviso that blocking moiet(ies)
present at the N- and/or C-termini of a compstatin analog may be
absent, replaced by a linker (which may comprise a blocking
moiety), or attached to a different N- or C-terminal amino acid
present in the corresponding variant(s).
[0261] Any compstatin analog, e.g., any compound comprising any of
SEQ ID NOs: 3-41 may be attached via its N-terminus or C-terminus
directly or indirectly to any moiety comprising a reactive
functional group, e.g., any of Formulas I-XVI or Compound I-III, in
various embodiments.
[0262] In some embodiments a CRM comprises a polypeptide that
occurs in human serum, or a fragment thereof or a substantially
similar variant of the polypeptide or fragment thereof. In some
embodiments the polypeptide, fragment, or variant has a molecular
weight of between 5 kD and 150 kD, e.g., at least 5, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100 kd, or more, e.g., between 100 and 120,
or 120 and 150 kD. In some embodiments, producing a long-acting
compstatin analog comprises reacting a compstatin analog comprising
a reactive functional group with one or more amino acid side chains
of the polypeptide, wherein the side chain comprises a compatible
functional group. In some embodiments, producing a long-acting
compstatin analog comprises reacting a compstatin analog comprising
a reactive functional group with the N-terminal amine and/or
C-terminal carboxyl group of the polypeptide. In some embodiments,
producing a long-acting compstatin analog comprises reacting a
compstatin analog comprising an amine-reactive functional group
with amino acids having a side chain comprising a primary amine
(e.g., lysine) and/or with the N-terminal amine of the polypeptide.
In some embodiments, producing a long-acting compstatin analog
comprises reacting a compstatin analog comprising a
carboxyl-reactive functional group with the C-terminal carboxyl
group of the polypeptide. In some embodiments a compstatin analog
moiety is attached at each terminus of the polypeptide and,
optionally, to the side chain of one or more internal amino acids.
In some embodiments, producing a long-acting compstatin analog
comprises reacting a compstatin analog comprising a
sulfhydryl-reactive functional group with one or more sulfhydryl
groups of the polypeptide.
[0263] In some embodiments, at least one reactive functional group
is introduced into the polypeptide. For example, in some
embodiments at least one side chain of the polypeptide is modified
to convert a first reactive functional group to a different
reactive functional group prior to reaction with the compstatin
analog. In some embodiments a thiol is introduced. Several methods
are available for introducing thiols into biomolecules, including
the reduction of intrinsic disulfides, as well as the conversion of
amine, aldehyde or carboxylic acid groups to thiol groups.
Disulfide crosslinks of cystines in proteins can be reduced to
cysteine residues by dithiothreitol (DTT),
tris-(2-carboxyethyl)phosphine (TCEP), or
tris-(2-cyanoethyl)phosphine Amines can be indirectly thiolated by
reaction with succinimidyl 3-(2-pyridyldithio)propionate (SPDP)
followed by reduction of the 3-(2-pyridyldithio)propionyl conjugate
with DTT or TCEP Amines can be indirectly thiolated by reaction
with succinimidyl acetylthioacetate followed by removal of the
acetyl group with 50 mM hydroxylamine or hydrazine at near-neutral
pH Amines can be directly thiolated by reaction with
2-iminothiolane, which preserve the overall charge of the molecule
and introduces a free thiol. Tryptophan residues in thiol-free
proteins can be oxidized to mercaptotryptophan residues, which can
then be modified by iodoacetamides or maleimides. A polypeptide
comprising one or more thiols may be reacted with a compstatin
analog comprising a maleimide group, such as
Ac-Ile-Cys*-Val-Trp(1-Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys*-Thr-AEEAc-Lys--
(C(.dbd.O)--(CH.sub.2).sub.5-Mal)-NH.sub.2 (SEQ ID NO: 58) to
generate a long-acting compstatin analog.
[0264] In some embodiments the polypeptide is recombinantly
produced. In some embodiments the polypeptide is at least in part
recombinantly produced (e.g., in bacteria or in eukaryotic host
cells such as fungal, insect, plant, or vertebrate) and/or at least
in part produced using chemical synthesis. In some embodiments the
polypeptide is purified. In some embodiments the polypeptide is
glycosylated. In some embodiments the polypeptide is
non-glycosylated. In some embodiments the polypeptide is human
serum albumin (HSA). In some embodiments a substantially similar
variant of the polypeptide is sufficiently similar to the
polypeptide of which it is a variant so as to not be recognized as
foreign by a normal immune system of a subject, e.g., a human
subject. In some embodiments alterations in the sequence of
substantially similar variant as compared with the polypeptide of
which it is a variant are selected so as to avoid generating MHC
Class I epitopes. Various methods known in the art can be used to
predict whether a sequence comprises an MHC Class I epitope.
[0265] The structure of compstatin is known in the art, and NMR
structures for a number of compstatin analogs having higher
activity than compstatin are also known (Malik, supra). Structural
information may be used to design compstatin mimetics. In some
embodiments, a compstatin mimetic is any compound that competes
with compstatin or any compstatin analog (e.g., a compstatin analog
whose sequence is set forth in Table 2) for binding to C3 or a
fragment thereof (such as a 40 kD fragment of the .beta. chain to
which compstatin binds). In some embodiments, the compstatin
mimetic has an activity equal to or greater than that of
compstatin. In some embodiments, the compstatin mimetic is more
stable, orally available, or has a better bioavailability than
compstatin. The compstatin mimetic may be a peptide, nucleic acid,
or small molecule. In certain embodiments the compstatin mimetic is
a compound that binds to the binding site of compstatin as
determined in a compstatin-C3 structure, e.g., a crystal structure
or a 3-D structure derived from NMR experiments. In certain
embodiments the compstatin mimetic is a compound that could
substitute for compstatin in a compstatin-C3 structure and would
form substantially the same intermolecular contacts with C3 as
compstatin. In certain embodiments the compstatin mimetic is a
compound that binds to the binding site of a peptide having a
sequence set forth in Table 2, e.g., SEQ ID NO: 14, 21, 28, 29, 32,
33, 34, or 36 or other compstatin analog sequence or in certain
embodiments SEQ ID NO: 30 or 31, in a peptide-C3 structure. In
certain embodiments the compstatin mimetic is a compound that could
substitute for a peptide having a sequence set forth in Table 2,
e.g., SEQ ID NO: 14, 21, 28, 29, 32, 33, 34, or 36 or other
compstatin analog sequence or in certain embodiments SEQ ID NO: 30
or 31, in a peptide-C3 structure and would form substantially the
same intermolecular contacts with C3 as the peptide. In certain
embodiments the compstatin mimetic has a non-peptide backbone but
has side chains arranged in a sequence designed based on the
sequence of compstatin.
[0266] One of skill in the art will appreciate that once a
particular desired conformation of a short peptide has been
ascertained, methods for designing a peptide or peptidomimetic to
fit that conformation are well known. See, e.g., G. R. Marshall
(1993), Tetrahedron, 49: 3547-3558; Hruby and Nikiforovich (1991),
in Molecular Conformation and Biological Interactions, P. Balaram
& S. Ramasehan, eds., Indian Acad. of Sci., Bangalore, P P.
429-455), Eguchi M, Kahn M., Mini Rev Med Chem., 2(5):447-62, 2002.
Of particular relevance to the present invention, the design of
peptide analogs may be further refined by considering the
contribution of various side chains of amino acid residues, e.g.,
for the effect of functional groups or for steric considerations as
described in the art for compstatin and analogs thereof, among
others.
[0267] It will be appreciated by those of skill in the art that a
peptide mimic may serve equally well as a peptide for the purpose
of providing the specific backbone conformation and side chain
functionalities required for binding to C3 and inhibiting
complement activation. Accordingly, it is contemplated as being
within the scope of the present invention to produce and utilize
C3-binding, complement-inhibiting compounds through the use of
either naturally-occurring amino acids, amino acid derivatives,
analogs or non-amino acid molecules capable of being joined to form
the appropriate backbone conformation. A non-peptide analog, or an
analog comprising peptide and non-peptide components, is sometimes
referred to herein as a "peptidomimetic" or "isosteric mimetic," to
designate substitutions or derivations of a peptide that possesses
much the same backbone conformational features and/or other
functionalities, so as to be sufficiently similar to the
exemplified peptides to inhibit complement activation. More
generally, a compstatin mimetic is any compound that would position
pharmacophores similarly to their positioning in compstatin, even
if the backbone differs.
[0268] The use of peptidomimetics for the development of
high-affinity peptide analogs is well known in the art. Assuming
rotational constraints similar to those of amino acid residues
within a peptide, analogs comprising non-amino acid moieties may be
analyzed, and their conformational motifs verified, by means of the
Ramachandran plot (Hruby & Nikiforovich 1991), among other
known techniques.
[0269] One of skill in the art will readily be able to establish
suitable screening assays to identify additional compstatin
mimetics and to select those having desired inhibitory activities.
For example, compstatin or an analog thereof could be labeled
(e.g., with a radioactive or fluorescent label) and contacted with
C3 in the presence of different concentrations of a test compound.
The ability of the test compound to diminish binding of the
compstatin analog to C3 is evaluated. A test compound that
significantly diminishes binding of the compstatin analog to C3 is
a candidate compstatin mimetic. For example, a test compound that
diminishes steady-state concentration of a compstatin analog-C3
complex, or that diminishes the rate of formation of a compstatin
analog-C3 complex by at least 25%, or by at least 50%, is a
candidate compstatin mimetic. One of skill in the art will
recognize that a number of variations of this screening assay may
be employed. Compounds to be screened include natural products,
libraries of aptamers, phage display libraries, compound libraries
synthesized using combinatorial chemistry, etc. The invention
encompasses synthesizing a combinatorial library of compounds based
upon the core sequence described above and screening the library to
identify compstatin mimetics. Any of these methods could also be
used to identify new compstatin analogs having higher inhibitory
activity than compstatin analogs tested thus far.
[0270] Other Compounds that Inhibit C3 Activation or Activity
[0271] Other compounds, e.g., polypeptides, small molecules,
monoclonal antibodies, aptamers, etc., that bind to C3 or C3a
receptors (C3aR) are of use in certain embodiments of the
invention. In certain embodiments the complement inhibitor
comprises an Efb protein from Staphylococcus aureus or a variant or
derivative or mimetic thereof that can bind to C3 and inhibit its
activation and/or bind to and inhibit C3b. Exemplary agents are
described in PCT Application Pub. WO/2004/094600. In certain
embodiments the complement inhibitor comprises a Staphylococcus
complement inhibitor (SCIN) protein from Staphylococcus aureus or a
variant or derivative or mimetic of such protein that can bind to
C3 convertase and inhibit its activation and/or bind to and inhibit
C3b. Aptamers that bind to and inhibit C3 may be identified using
methods such as SELEX. U.S. Pat. Pub. No. 20030191084 discloses
aptamers that bind to C1q, C3 and C5.
[0272] In some embodiments, a protease that degrades C3 may be used
as a complement inhibitor. For example, U.S. Pat. No. 6,676,943
discloses human complement C3-degrading protein from Streptococcus
pneumoniae. Such proteins, or variants thereof, may be used in
certain embodiments of the invention.
[0273] U.S. Pat. No. 5,942,405, PCT/IB2006/002557
(WO/2007/034277-ARYL SUBSTITUTED IMIDAZO [4,5-C] PYRIDINE COMPOUNDS
AS C3A RECEPTOR ANTAGONISTS); PCT/IB2006/002568
(WO/2007/034282-DIARYL-IMIDAZOLE COMPOUNDS CONDENSED WITH A
HETEROCYCLE AS C3A RECEPTOR ANTAGONISTS) PCT/IB2006/002561
(WO2007034278--FUSED IMIDAZOLE DERIVATIVES AS C3A RECEPTOR
ANTAGONISTS) PCT/US2007/026237 (WO2008079371) MODULATORS OF C3A
RECEPTOR AND METHODS OF USE THEREOF disclose exemplary C3aR
antagonists. In some embodiments, an RNAi agent that inhibits
expression of C3 or C3aR may be used.
[0274] Compounds that Inhibit Factor B Activation or Activity
[0275] In certain embodiments a complement inhibitor inhibits
activation or activity of factor B. For example, the complement
inhibitor may bind to factor B and, e.g., inhibit activation of
factor B. Exemplary agents that inhibit activation or activity of
factor B include, e.g., antibodies, antibody fragments, peptides,
small molecules, and aptamers. Exemplary antibodies that inhibit
factor B are described in U.S. Pat. Pub. No. 20050260198. In
certain embodiments an antibody or antigen-binding fragment
selectively binds to factor B within the third short consensus
repeat (SCR) domain. In certain embodiments the antibody prevents
formation of a C3bBb complex. In certain embodiments the antibody
or antigen-binding fragment prevents or inhibits cleavage of factor
B by factor D. In some embodiments, an antibody binds to the Bb
portion of factor B. PCT/US2008/074489 (WO/2009/029669) discloses
exemplary antibodies, e.g., the antibody produced by the hybridoma
clone deposited under ATCC Accession Number PTA-8543. In some
embodiments, a humanized version of said antibody is used, which
may be an antibody fragment. In certain embodiments a complement
inhibitor, e.g., antibody, small molecule, aptamer, polypeptide, or
peptide, binds to substantially the same binding site on factor B
as an antibody described in U.S. Pat. Pub. No. 20050260198 or
WO/2009/029669. In some embodiments, the complement inhibitor
comprises the monoclonal antibody fragment known as TA106 (formerly
under development by Taligen Therapeutics), or antibody, small
molecule, aptamer, polypeptide, or peptide, binds to substantially
the same binding site on factor B as TA106 is used. In some
embodiments, a peptide that binds to and inhibits factor B is
identified using, for example, a method such as phage display. In
some embodiments, a complement inhibitor comprises an aptamer that
binds to and inhibits factor B. In some embodiments, an RNAi agent
that inhibits expression of factor B may be used.
[0276] Compounds that Inhibit Factor D Activity
[0277] In certain embodiments the complement inhibitor inhibits
factor D. For example, the complement inhibitor may bind to factor
D. Exemplary agents include antibodies, antibody fragments,
peptides, small molecules, and aptamers. Exemplary antibodies that
inhibit factor D are described in U.S. Pat. No. 7,112,327. In
certain embodiments the complement inhibitor is an antibody, small
molecule, aptamer, or polypeptide that binds to substantially the
same binding site on factor D as an antibody described in U.S. Pat.
No. 7,112,327. FCFD4514S (formerly under development by Tanox as
TNX-234), is a humanized monoclonal antibody fragment that binds
Factor D. In certain embodiments the complement inhibitor comprises
FCFD4514S or an antibody, small molecule, aptamer, or polypeptide
that binds to substantially the same binding site on factor D as
FCFD4514S. Exemplary polypeptides that inhibit alternative pathway
activation and are believed to inhibit factor D are disclosed in
U.S. Pub. No. 20040038869. Use of peptides that bind to and inhibit
factor D, which may be identified using methods such as phage
display, is within the scope of the invention. Use of aptamers that
bind to and inhibit factor D, which may be identified using methods
such as SELEX, is within the scope of the invention. In some
embodiments, an RNAi agent that inhibits expression of factor D may
be used.
[0278] Mammalian Complement Regulatory Proteins and Complement
Receptors
[0279] In some embodiments the complement inhibitor comprises at
least a portion of a mammalian, e.g., human, complement regulatory
protein or complement receptor. Examples of complement regulatory
proteins include, e.g., CFH, CFH related proteins (such as CFHR1),
CFI, CR1, DAF, MCP, CD59, C4 bp, and complement receptor 2
inhibitor trispanning (CRIT; Inal, J., et al, J Immunol.,
174(1):356-66, 2005). In some embodiments the complement regulatory
polypeptide is one that is normally membrane-bound in its naturally
occurring state. In some embodiments of the invention a fragment of
such polypeptide that lacks some or all of a transmembrane and/or
intracellular domain is used. Soluble forms of complement receptor
1 (sCR1), or soluble portions of other complement receptors, for
example, are of use in certain embodiments. For example the
compounds known as TP10 or TP20 (Avant Therapeutics) can be used.
In some embodiments a soluble complement control protein, e.g., CFH
or a CFH related protein, is used. In some embodiments the
complement inhibitor is a C3b/C4b Complement Receptor-like molecule
such as those described in U.S. Pat. Pub. No. 20020192758. Variants
and fragments of mammalian complement regulatory proteins or
receptors that retain complement inhibiting activity can be used in
certain embodiments.
[0280] Chimeric Complement Inhibitors
[0281] In certain embodiments of the invention the complement
inhibitor comprises a chimeric polypeptide comprising a first
polypeptide that inhibits complement activation, linked, e.g.,
covalently linked, to a second polypeptide that inhibits complement
activation and/or that binds to a complement component or
complement activation product. In some embodiments, at least one of
the polypeptides comprises at least a portion of a mammalian
complement regulatory protein. The chimeric polypeptide may contain
one or more additional domains located, e.g., between the first and
second polypeptides or at a terminus. For example, the first and
second polypeptides can be separated by a spacer polypeptide.
[0282] In some embodiments, the first and second polypeptides each
comprise at least a portion of a mammalian complement regulatory
protein. In some embodiments complement inhibitor comprises at
least a portion of DAF and at least a portion of MCP. Exemplary
chimeric polypeptides are disclosed, e.g., in U.S. Pat. No.
5,679,546, e.g., CAB-2 (also known as MLN-2222). In some
embodiments the polypeptide comprises at least 4 SCR domains of at
least one mammalian complement regulatory protein or complement
receptor. In some embodiments the polypeptide comprises at least 4
SCR domains of each of first and second distinct mammalian
complement regulatory proteins.
[0283] In some embodiments, a chimeric polypeptide comprises at
least a portion of complement receptor 1 (CR1), complement receptor
2 (CR2), complement receptor 3 (CR3), complement receptor 4 (CR4)
or a variant or fragment of CR1, CR2, CR3, or CR4 that binds to one
or more complement components or complement activation products
such as C3b, iC3b, C3d, and/or C3dg. In some embodiments, the
polypeptide comprises at least 4 SCRs, e.g., at least 4 SCRs of CR1
or CR2. For example, the polypeptide can comprise the 4 N-terminal
SCRs of CR2 (e.g., residues 1-250 of the mature protein). In some
embodiments the chimeric polypeptide comprises at least 4 SCR
domains of a mammalian complement regulatory protein and at least 4
SCR domains of a mammalian complement receptor.
[0284] Compounds that Inhibit Properdin
[0285] In some embodiments of the invention antiproperdin
antibodies, antibody fragment, or other anti-properdin agents are
used. See, e.g., U.S. Pat. Pub. No. 20030198636 or
PCT/US2008/068530 (WO/2009/110918-ANTI-PROPERDIN ANTIBODIES) for
examples.
[0286] Compounds that Inhibit Components of Lectin Pathway
[0287] In some embodiments the compounds inhibit one or more
components of the lectin pathway. See, e.g., WO/2007/117996)
METHODS FOR TREATING CONDITIONS ASSOCIATED WITH MASP-2 DEPENDENT
COMPLEMENT ACTIVATION.
[0288] Compounds that Inhibit C5 Activation or Activity
[0289] In certain embodiments the complement inhibitor inhibits
activation of C5. For example, the complement inhibitor may bind to
C5 and inhibit its cleavage. In some embodiments, the complement
inhibitor inhibits physical interaction of C5 with C5 convertase
by, e.g., binding to C5 or C5 convertase or to C5 at a site that
would ordinarily participate in such physical interaction.
Exemplary agents that inhibit C5 activation include antibodies,
antibody fragments, polypeptides, small molecules, and aptamers.
Exemplary compounds, e.g., antibodies, that bind to C5 are
described, for example, in U.S. Pat. No. 6,534,058; PCT/US95/05688
(WO 1995/029697), PCT/EP2010/007197 (WO2011063980); U.S. Pat. Pub.
No. 20050090448; and U.S. Pat. Pub. No. 20060115476. U.S. Pat. Pub.
No. 20060105980 discloses aptamers that bind to and inhibit C5. In
some embodiments, a humanized anti-C5 monoclonal antibody, e.g.,
eculizumab (also known as h5G1.1-mAb; Soliris.RTM.) (Alexion), or a
fragment or derivative thereof that binds to C5. In some
embodiments, an antibody comprising at least some of the same
complementarity determining regions (CDR1, CDR2 and/or CDR3), e.g.,
all of CDR1, CDR2, and CDR3, as those of eculizumab's heavy chain
and/or light chain is used. In some embodiments, the antibody
comprises at least some of the same framework regions as
eculizumab. In some embodiments, an antibody that binds to
substantially the same binding site on C5 as eculizumab is used. In
some embodiments, pexelizumab (also known as h5G1.1-scFv), a
humanized, recombinant, single-chain antibody derived from
h5G1.1-mAb, is used. In certain embodiments the complement
inhibitor comprises a Staphylococcus SSL7 protein from
Staphylococcus aureus or a variant or derivative or mimetic of such
protein that can bind to C5 and inhibit its cleavage.
[0290] As noted above, bispecific or multispecific antibodies can
be used. For example, PCT/US2010/039448 (WO/2010/151526) discloses
bispecific antibodies described as binding to two or more different
proteins, wherein at least two of the proteins are selected from
C5a, C5b, a cellular receptor for C5a (e.g., C5aR1 or C5L2), the
C5b-9 complex, and a component or intermediate of terminal
complement such as C5b-6, C5b-7, or C5b-8. In some embodiments an
RNAi agent that inhibits expression of C5 or C5aR may be used.
[0291] In some embodiments, a complement inhibitor known as OmCI,
or a variant, derivative, or mimetic thereof, is used. OmCI binds
to C5 and inhibits its activation most likely by inhibiting
interaction with convertase. OmCI is naturally produced by the tick
Ornithodoros moubata. See, e.g., PCT/GB2004/002341 (WO/2004/106369)
and PCT/GB2010/000213 (WO/2010/100396), for description of OmCI and
certain variants thereof. It has been shown that OmCI binds to
eicosanoids, in particular leukotriene (LKs), e.g., LTB4. In some
embodiments, an OmCI polypeptide (or a variant, derivative, or
fragment thereof) that retains the capacity to binds to a LK, e.g.,
LTB4, is used. In some embodiments, an OmCI polypeptide (or a
variant, derivative, or fragment thereof) that has reduced capacity
or substantially lacks capacity to bind to a LK, e.g., LTB4, is
used.
[0292] In some embodiments the agent is an antagonist of a C5a
receptor (C5aR). In some embodiments, the C5aR antagonist comprises
a peptide. Exemplary C5a receptor antagonists include a variety of
small cyclic or acyclic peptides such as those described in March,
D R, et al., Mol. Pharmacol., 65(4), 2004, and in Woodruff, T M, et
al., J Pharmacol Exp Ther., 314(2):811-7, 2005, U.S. Pat. No.
6,821,950; U.S. Ser. No. 11/375,587; and/or PCT/US06/08960
(WO2006/099330), or a mimetic thereof. In certain embodiments the
complement inhibitor binds to C5aR and inhibits binding of C5a
thereto. In certain embodiments a cyclic peptide comprising the
sequence [OPdChaWR] (SEQ ID NO: 59) is used. In certain embodiments
a cyclic peptide comprising the sequence [KPdChaWR] (SEQ ID NO: 60)
is used. In certain embodiments a peptide comprising the sequence
(Xaa).sub.n[OPdChaWR] (SEQ ID NO: 61) is used, wherein Xaa is an
amino acid residue and n is between 1 and 5. In certain embodiments
a peptide comprising the sequence (Xaa).sub.n[KPdChaWR] (SEQ ID NO:
62) is used, wherein Xaa is an amino acid residue and n is between
1 and 5. In certain embodiments n is 1. In certain embodiments n is
1 and Xaa is a standard or nonstandard aromatic amino acid. For
example, the peptides F-[OPdChaWR] (SEQ ID NO: 63), F-[KPdChaWR]
(SEQ ID NO: 64); Cin-[OPdChaWR] (SEQ ID NO: 65), and
HCin-[OPdChaWR] (SEQ ID NO: 66) are of use in certain embodiments.
Optionally the free terminus comprises a blocking moiety, e.g., the
terminal amino acid is acetylated. For example, in some embodiments
the C5aR antagonist is AcF-[OPdChaWR] (SEQ ID NO: 67) (also known
as PMX-53). (Abbreviations: 0: ornithine; Cha: cyclohexylalanine;
Cin: cinnamoyl; Hcin: hydrocinnamoyl; square brackets denote
internal peptide bond). In some embodiments, a C5aR antagonist
comprises a compound, e.g., a peptide, disclosed in U.S. Pat. Pub.
No. 20060183883 (U.S. Ser. No. 10/564,788), e.g., a compound as
represented therein by formula I, formula II, formula IV, formula
V, or formula VI. An exemplary C5aR antagonist is the peptide known
as JPE-1375 (Jerini AG, Germany)
[0293] In some embodiments, a C5aR antagonist is a small molecule.
Various small molecule C5aR antagonists are disclosed in the
following references: PCT/US2005/015897 (WO/2005/110416;
4,5-DISUBSTITUTED-2-ARYL PYRIMIDINES); PCT/EP2006/005141
(WO2006128670); PCT/US2008/072902 (WO/2009/023669; SUBSTITUTED
5,6,7,8-TETRAHYDROQUINOLINE DERIVATIVES); PCT/US2009/068941
(WO/2010/075257; C5AR ANTAGONISTS). An exemplary small molecule
C5aR antagonist is CCX168 (ChemoCentryx, Mountain View,
Calif.).
[0294] In certain embodiments the complement inhibitor is an agent,
e.g., an antibody, small molecule, aptamer, or polypeptide, that
binds to substantially the same binding site on C5 or C5aR as a
compound described in any of the afore-mentioned references
disclosing agents that bind to C5 or C5aR. In some embodiments the
complement inhibitor is not an antagonist of a C5a receptor.
[0295] Multimodal Complement Inhibitors
[0296] In certain embodiments of the invention the complement
inhibitor binds to more than one complement protein and/or inhibits
more than one step in a complement activation pathway. Such
complement inhibitors are referred to herein as "multimodal". In
certain embodiments of the invention the complement inhibitor
comprises a virus complement control protein (VCCP). The invention
contemplates use of any of the agents described in U.S. Ser. No.
11/247,886 and PCT/US2005/36547. Poxviruses and herpes viruses are
families of large, complex viruses with a linear double-stranded
DNA genome. Certain of these viruses encode immunomodulatory
proteins that are believed to play a role in pathogenesis by
subverting one or more aspects of the normal immune response and/or
fostering development of a more favorable environment in the host
organism (Kotwal, G J, Immunology Today, 21(5), 242-248, 2000).
Among these are VCCPs. Poxvirus complement control proteins are
members of the complement control protein (CCP) superfamily and
typically contain 4 SCR modules. In certain embodiments the VCCP is
a poxvirus complement control protein (PVCCP). The PVCCP can
comprise a sequence encoded by, e.g., vaccinia virus, variola major
virus, variola minor virus, cowpox virus, monkeypox virus,
ectromelia virus, rabbitpox virus, myxoma virus, Yaba-like disease
virus, or swinepox virus. In other embodiments the VCCP is a
herpesvirus complement control protein (HVCCP). The HVCCP can
comprise a sequence encoded by a Macaca fuscata rhadinovirus,
cercopithecine herpesvirus 17, or human herpes virus 8. In other
embodiments the HVCCP comprises a sequence encoded by herpes
simplex virus saimiri ORF 4 or ORF 15 (Albrecht, J C. &
Fleckenstein, B., J. Virol., 66, 3937-3940, 1992; Albrecht, J., et
al., Virology, 190, 527-530, 1992).
[0297] The VCCP may inhibit the classical complement pathway, the
alternate complement pathway, the lectin pathway, or any two or
more of these. In certain embodiments of the invention the VCCP,
e.g., a PVCCP, binds to C3b, C4b, or both. In certain embodiments
of the invention the PVCCP comprises one or more putative heparin
binding sites (K/R-X-K/R) and/or possesses an overall positive
charge. In some embodiments the PVCCP comprises at least 3 SCR
modules (e.g., modules 1-3), e.g., 4 SCR modules. The PVCCP protein
can be a precursor of a mature PVCCP (i.e., can include a signal
sequence that is normally cleaved off when the protein is expressed
in virus-infected cells) or can be a mature form (i.e., lacking the
signal sequence).
[0298] Vaccinia complement control protein (VCP) is a virus-encoded
protein secreted from vaccinia infected cells. VCP is 244 amino
acids in length, contains 4 SCRs, and is naturally produced by
intracellular cleavage of a 263 amino acid precursor. VCP runs as
an .about.35 kD protein in a 12% SDS/polyacrylamide gel under
reducing conditions and has a predicted molecular mass of about
28.6 kD. VCP is described in U.S. Pat. Nos. 5,157,110 and
6,140,472, and in Kotwal, G K, et al., Nature, 355, 176-178, 1988.
FIGS. 3A and 3B of U.S. Ser. No. 11/247,886 and PCT/US2005/36547
(WO2006042252) show the sequence of the precursor and mature VCP
proteins, respectively. VCP has been shown to inhibit the classical
pathway of complement activation via its ability to bind to C3 and
C4 and act as a cofactor for factor I mediated cleavage of these
components as well as promoting decay of existing convertase
(Kotwal, G K, et al., Science, 250, 827-830, 1990; McKenzie et al.,
J. Infect. Dis., 1566, 1245-1250, 1992). It has also been shown to
inhibit the alternative pathway by causing cleavage of C3b into
iC3b and thereby preventing the formation of the alternative
pathway C3 convertase (Sahu, A, et al., J. Immunol., 160,
5596-5604, 1998). VCP thus blocks complement activation at multiple
steps and reduces levels of the proinflammatory chemotactic factors
C3a, C4a, and C5a.
[0299] VCP also possesses the ability to strongly bind heparin in
addition to heparan sulfate proteoglycans. VCP contains two
putative heparin binding sites located in modules 1 and 4 (Jha, P
and Kotwal, G J, and references therein). VCP is able to bind to
the surface of endothelial cells, possibly via interaction with
heparin and/or heparan sulfate at the cell surface, resulting in
decreased antibody binding (Smith, S A, et al., J. Virol., 74(12),
5659-5666, 2000). VCP can be taken up by mast cells and possibly
persist in tissue for lengthy periods of time, thereby potentially
prolonging its activity (Kotwal, G J, et al., In GP. Talwat, et al.
(eds), 10.sup.th International Congress of Immunology., Monduzzi
Editore, Bologna, Italy, 1998). In addition, VCP can reduce
chemotactic migration of leukocytes by blocking chemokine binding
(Reynolds, D, et al., in S. Jameel and L. Villareal (ed., Advances
in animal virology. Oxford and IBN Publishing, New Delhi, India,
1999). VCP and other PVCCPs have a relatively small size relative
to mammalian CCPs, which is advantageous for delivery in the
present invention.
[0300] Variola virus major and minor encode proteins that are
highly homologous to VCP and are referred to as smallpox inhibitor
of complement enzymes (SPICE) (Rosengard, A M, et al., Proc. Natl.
Acad. Sci., 99(13), 8803-8813. U.S. Pat. No. 6,551,595). SPICE from
various variola strains sequenced to date differs from VCP by about
5% (e.g., about 11 amino acid differences). Similarly to VCP, SPICE
binds to C3b and C4b and causes their degradation, acting as a
cofactor for factor I. However, SPICE degrades C3b approximately
100 times as fast as VCP and degrades C4b approximately 6 times as
fast as VCP. The amino acid sequence of SPICE is presented in FIG.
6 (SEQ ID NO: 12) of U.S. Ser. No. 11/247,886 and PCT/US2005/36547
(WO2006042252) and can be described as follows. Referring to FIG. 6
of U.S. Ser. No. 11/247,886 and PCT/US2005/36547 (WO2006042252), a
signal sequence extends from amino acid 1 to about amino acid 19.
Four SCRs extend from about amino acid 20 to amino acid 263. Each
SCR is characterized by four cysteine residues. The four cysteine
residues form two disulfide bonds in the expressed protein. The
boundaries of each SCR are best defined by the first and fourth
cysteine residues in the sequence that forms the disulfide bonds of
the SCR. An invariant tryptophan residue is present between
cysteine 3 and cysteine 4 of each SCR. SCR1 extends from amino acid
20 or 21 to amino acid 81. Both residues are cysteines that may be
involved in disulfide bonding. SCR2 extends from amino acid 86 to
amino acid 143. SCR3 extends from amino acid 148 to amino acid 201.
SCR4 extends from amino acid 206 to amino acid 261. The SCRs
include the complement binding locations of SPICE. SPICE or any of
the portions thereof that inhibit complement activation, e.g.,
SPICE and SPICE-related polypeptides containing four SCRs, such as
those described in U.S. Pat. No. 6,551,595, are of use in the
present invention.
[0301] Complement control proteins from cowpox virus (referred to
as inflammation modulatory protein, IMP) and monkeypox virus
(referred to herein as monkeypox virus complement control protein,
MCP) have also been identified and sequenced (Miller, C G, et al.,
Virology, 229, 126-133, 1997 and Uvarova, E A and Shchelkunov, S N,
Virus Res., 81(1-2), 39-45, 2001). MCP differs from the other
PVCCPs described herein in that it contains a truncation of the
C-terminal portion of the fourth SCR.
[0302] It will be appreciated that the exact sequence of complement
control proteins identified in different virus isolates may differ
slightly. Such proteins fall within the scope of the present
invention. Complement control proteins from any such isolate may be
used, provided that the protein has not undergone a mutation that
substantially abolishes its activity. Thus the sequence of a VCCP
such as SPICE or VCP may differ from the exact sequences presented
herein or under the accession numbers listed in Table 3. It will
also be appreciated that a number of amino acid alterations, e.g.,
additions, deletions, or substitutions such as conservative amino
acid substitutions, may be made in a typical polypeptide such as a
VCCP without significantly affecting its activity, such that the
resulting protein is considered equivalent to the original
polypeptide. The viral polypeptides identified by accession number
in Table 3 below are of use in various embodiments of the
invention.
TABLE-US-00009 TABLE 3 Representative Viral Complement Control
Proteins Virus Protein Accession Virus Type Variola D12L NP_042056
Orthopoxvirus D15L (SPICE) AAA69423 Orthopoxvirus Vaccinia VCP
AAO89304 Orthopoxvirus Cowpox CPXV034 AAM13481 Orthopoxvirus C17L
CAA64102 Orthopoxvirus Monkeypox D14L AAV84857 Orthopoxvirus
Ectromelia virus Complement control protein CAE00484 Orthopoxvirus
Rabbitpox RPXV017 AAS49730 Orthopoxvirus Macaca fuscata
rhadinovirus JM4 AAS99981 Rhadinavirus (Herpesvirus) Cercopithecine
herpesvirus 17 Complement binding NP_570746 Herpesvirus protein
(ORF4) Human herpes virus 8 Complement binding AAB62602 Herpesvirus
protein (ORF4)
[0303] In addition to the VCCPs described above, a number of other
viral proteins exist that interfere with one or more steps in a
complement pathway. These proteins are also of use in certain
embodiments of the present invention. Certain of these proteins do
not necessarily display clear homology to cellular complement
regulators known to date. For example, HSV-1, HSV-2, VZV, PRV,
BHV-1, EHV-1, and EHV-4 all encode versions of a conserved
glycoprotein known as gC (Schreurs, et al., J Virol., 62,
2251-2257, 1988; Mettenleiter, et al, J. Virol., 64, 278-286; 1990;
Herold, et al., J Virol., 65, 1090-1098; 1991). With the exception
of VZV, the gC protein encoded by these viruses binds to C3b
(Friedman, et al., Nature, 309, 633-634,1984; Huemer, et al., Virus
Res., 23, 271-280, 1993) gC1 (from HSV-1) accelerates decay of the
classical pathway C3 convertase and inhibits binding of properdin
and C5 to C3. Purified EBV virions possess an activity that
accelerates decay of the alternative pathway C3 convertase and
serves as a cofactor for the complement regulatory protein factor 1
(Mold et al., J Exp Med, 168, 949-969, 1988). The foregoing
proteins are referred to collectively as virus complement
interfering proteins (VCIPs). By any of a variety of means, such as
interfering with one or more steps of complement activation,
accelerating decay of a complement component, and/or enhancing
activity of a complement regulatory protein, these VCIPs are said
to inhibit complement. Any of these proteins, or derivatives
thereof, e.g., fragments or variants thereof, can be used as a
therapeutic agent in the invention. As in the case of VCCPs, will
be appreciated that the exact sequence of VCIPs identified in
different virus isolates may differ slightly. Such proteins fall
within the scope of the present invention.
[0304] In certain embodiments of the invention a fragment or
variant of a VCCP or VCIP is locally administered to a subject.
Preferred fragments and variants of a PVCCP possess at least one of
the following activities: (i) ability to bind to C3, C3b, or both;
(ii) ability to act as a cofactor for factor I cleavage of C3;
(iii) ability to bind to C4, C4b, or both; (iv) ability to act as a
cofactor for factor I cleavage of C4; (v) ability to accelerate
decay of existing C3 convertase of the classical pathway, alternate
pathway, or both; (vi) ability to bind heparin; (vii) ability to
bind to heparan sulfate proteoglycans; (viii) ability to reduce
chemotactic migration of leukocytes; (ix) ability to block
chemokine (e.g, MIP-1.alpha.) binding, e.g., to the surface of a
cell (e.g., a leukocyte or endothelial cell surface); (x) ability
to inhibit antibody binding to class I MHC molecules; (xi) ability
to inhibit the classical complement pathway; (xii) ability to
inhibit the alternative complement pathway; and (xiii) ability to
inhibit complement-mediated cell lysis. Preferred PVCCP fragments
and variants display complement binding activity, by which is meant
ability to detectably bind to one or more complement components,
preferably (in the case of VCCPs) selected from the group
consisting of: C3, C3b, C4, and C4b. Preferred fragments or
variants of HVCCPs may also display ability to detectably bind to
one or more complement components. Preferably the binding of the
VCCP to the complement component is specific. It will be understood
that a VCCP may be able to bind to only a single complement
component or may be able to bind to more than one different
complement component.
[0305] In certain embodiments of the invention the PVCCP fragment
or variant comprises at least 3 SCR modules (e.g., modules 1-3),
preferably 4 SCR modules. Preferably each of the SCR modules
displays significant sequence identity to an SCR module found in a
naturally occurring PVCCP, e.g., VCP or SPICE. Preferably the
multiple SCR modules are arranged in an N to C manner so as to
maximize overall identity to a naturally occurring PVCCP. If the
sequence of a PVCCP fragment or variant contains an SCR domain that
differs from the SCR consensus sequence at one or more positions,
in certain embodiments of the invention the amino acid(s) at the
one or more differing positions is identical to that found at a
corresponding position in the most closely related SCR found in a
naturally occurring PVCCP. In certain embodiments the PVCCP variant
comprises at least one SCR module from a first PVCPP and at least
one SCR module from a second PVCPP. In certain embodiments the
PVCCP variant comprises at least one SCR module from a PVCCP and at
least one SCR from a mammalian complement control protein (RCA
protein). Any number of SCR modules, e.g., 1, 2, 3, 4, or more can
come from any particular PVCCP or RCA protein in various
embodiments of the invention. All such combinations and
permutations are contemplated, even if not explicitly set forth
herein.
[0306] Generally a fragment or variant of a naturally occurring
VCCP or VCIP possesses sufficient structural similarity to its
naturally occurring counterpart that it is recognized by a
polyclonal antibody that recognizes the naturally occurring
counterpart. In certain embodiments of the invention a fragment or
variant of a VCCP possesses sufficient structural similarity to VCP
or SPICE so that when its 3-dimensional structure (either actual or
predicted structure) is superimposed on the structure of VCP or
SPICE, the volume of overlap is at least 70%, preferably at least
80%, more preferably at least 90% of the total volume of the VCP
structure. A partial or complete 3-dimensional structure of the
fragment or variant may be determined by crystallizing the protein
as described for VCP (Murthy, 2001). Alternately, an NMR solution
structure can be generated, as performed for various VCP fragments
(Wiles, A P, et al., J. Mol. Biol. 272, 253-265, 1997). A modeling
program such as MODELER (Sali, A. and Blundell, T L, J. Mol. Biol.,
234, 779-815, 1993), or any other modeling program, can be used to
generate a predicted structure. The model can be based on the VCP
structure and/or any known SCR structure. The PROSPECT-PSPP suite
of programs can be used (Guo, J T, et al., Nucleic Acids Res.
32(Web Server issue):W522-5, Jul. 1, 2004). Similar methods may be
used to generate a structure for SPICE.
[0307] Fragments or variants of a VCCP or VCIP may be generated by
any available means, a large number of which are known in the art.
For example, VCCPs, VCIPs, and fragments or variants thereof can be
produced using recombinant DNA technology as described below. A
VCCP or VCIP fragment may be chemically synthesized, produced using
PCR amplification from a cloned VCCP or VCIP sequence, generated by
a restriction digest, etc. Sequences for a VCCP variant may be
generated by random mutagenesis of a VCCP sequence (e.g., using
X-rays, chemical agents, or PCR-based mutagenesis), site-directed
mutagenesis (e.g., using PCR or oligonucleotide-directed
mutagenesis, etc. Selected amino acids can be changed or added.
[0308] While not wishing to be bound by any theory, it is likely
that amino acid differences between naturally occurring PVCCPs
occur at positions that are relevant in conferring differences in
particular properties such as ability to bind heparin, activity
level, etc. For example, VCP and SPICE differ at only 11 amino
acids, but SPICE has a much higher activity as a cofactor for
cleavage of C3b (e.g., cleavage occurs at a much faster rate with
SPICE than with VCP). The amino acid differences are likely to be
responsible for the differential activities of the two proteins.
The amino acids at these positions are attractive candidates for
alteration to identify variants that have yet greater activity.
[0309] Additional Complement Inhibitors
[0310] In some embodiments a complement inhibitor is a naturally
occurring mammalian complement regulatory protein or a fragment or
derivative thereof. For example, the complement regulatory protein
may be CR1, DAF, MCP, CFH, or CFI. In some embodiments of the
invention the complement regulatory polypeptide is one that is
normally membrane-bound in its naturally occurring state. In some
embodiments of the invention a fragment of such polypeptide that
lacks some or all of a transmembrane and/or intracellular domain is
used. Soluble forms of complement receptor 1 (sCR1), for example,
are of use in the invention. For example the compounds known as
TP10 or TP20 (Avant Therapeutics) can be used. C1 inhibitor
(C1-INH) is also of use. In some embodiments a soluble complement
control protein, e.g., CFH, is used. In some embodiments of the
invention the polypeptide is modified to increase its
solubility.
[0311] In some embodiments, a complement inhibitor is a C1s
inhibitor. For example, U.S. Pat. No. 6,515,002 describes compounds
(furanyl and thienyl amidines, heterocyclic amidines, and
guanidines) that inhibit C1s. U.S. Pat. Nos. 6,515,002 and
7,138,530 describe heterocyclic amidines that inhibit C1s. U.S.
Pat. No. 7,049,282 describes peptides that inhibit classical
pathway activation. Certain of the peptides comprise or consist of
WESNGQPENN (SEQ ID NO: 68) or KTISKAKGQPREPQVYT (SEQ ID NO: 69) or
a peptide having significant sequence identity and/or
three-dimensional structural similarity thereto. In some
embodiments these peptides are identical or substantially identical
to a portion of an IgG or IgM molecule. U.S. Pat. No. 7,041,796
discloses C3b/C4b Complement Receptor-like molecules and uses
thereof to inhibit complement activation. U.S. Pat. No. 6,998,468
discloses anti-C2/C2a inhibitors of complement activation. U.S.
Pat. No. 6,676,943 discloses human complement C3-degrading protein
from Streptococcus pneumoniae.
V. Anti-Th17 Agents
[0312] An anti-Th17 agent is any agent that inhibits formation,
survival, and/or activity of Th17 cells or that inhibits production
or a biological activity of a Th17 cell effector molecule such as
IL-17. In some embodiments an anti-Th17 agent inhibits development,
proliferation, survival, and/or maturation of Th17 cells. In some
embodiments, an anti-Th17 agent inhibits production and/or a
biological activity of IL-6, IL-21, IL-23, and/or IL-113. In some
embodiments, an anti-Th17 agent inhibits production and/or activity
of a Th17 effector cytokine, e.g., IL-17A, IL-17F, IL-21, and/or
IL-22. Exemplary anti-Th17 agents include, e.g., agents that bind
to a Th17-associated cytokine or agents that bind to a receptor for
a Th17-associated cytokine and, e.g., block interaction of the
receptor with the endogenous cytokine but do not themselves
significantly activate the receptor. Exemplary anti-Th17 agents
include, e.g., antibodies, aptamers, soluble receptor fragments
(e.g., soluble extracellular domain of the relevant cytokine
receptor) or other polypeptides, peptides, small molecules, etc. In
some embodiments, an anti-Th17 agent comprises an antibody that
substantially lacks the capacity to activate complement. For
example, the antibody may have less than 10%, less than 5%, or less
than 1% complement stimulating activity as compared with full
length human IgG1. In some embodiments, the antibody comprises a
CH2 domain that has reduced ability to bind C1q as compared with
human IgG1 CH2 domain. In some embodiments, the antibody contains
CH1, CH2, and/or CH3 domains from human IgG4 and/or does not
contain CH1, CH2, and/or CH3 domains from human IgG1.
[0313] In some embodiments, an anti-Th17 agent has a molecular
weight of 1 kD or less. In some embodiments, an anti-Th17 agent has
a molecular weight between 1 kD and 2 kD, between 2 kD and 5 kD,
between 5 kD and 10 kD, between 10 kD and 20 kD, between 20 kD and
30 kD, between 30 kD and 50 kD, between 50 kD and 100 kD, or
between 100 kD and 200 kD.
[0314] In some embodiments an anti-Th17 agent comprises an
adnectin, affibody, anticalin, or other type of polypeptide
sometimes used in the art in lieu of an antibody, wherein the
polypeptide binds to a Th17-associated cytokine or cytokine
receptor.
[0315] A variety of anti-Th17 agents, e.g., agents that inhibit one
or more Th17-associated cytokines, are known in the art and may be
used in various embodiments.
[0316] Sequences of polypeptides of interest herein, e.g.,
Th17-associated cytokines and their receptors, are well known in
the art and available in public databases such as those available
through Entrez at the National Center for Biotechnology Information
(www.ncbi.nih.gov) or Universal Protein Resource (www.uniprot.org).
Exemplary databases include, e.g., GenBank, RefSeq, Gene, Protein,
Nucleotide, UniProtKB/SwissProt, UniProtKB/Trembl, and the like. In
general, sequences, e.g., mRNA and polypeptide sequences, in the
NCBI Reference Sequence database may be used as gene product
sequences for a gene of interest. Such sequences may be used, e.g.,
to produce a polypeptide useful as an antigen or reagent for
production, isolation, or characterization of an agent that binds
to the gene product. It will be appreciated that multiple alleles
of a gene may exist among individuals of the same species. For
example, differences in one or more nucleotides (e.g., up to about
1%, 2%, 3-5% of the nucleotides) of the nucleic acids encoding a
particular protein may exist among individuals of a given species.
Due to the degeneracy of the genetic code, such variations often do
not alter the encoded amino acid sequence, although DNA
polymorphisms that lead to changes in the sequence of the encoded
proteins can exist. Examples of polymorphic variants can be found
in, e.g., the Single Nucleotide Polymorphism Database (dbSNP),
available at the NCBI website at
www.ncbi.nlm.nih.gov/projects/SNP/. (Sherry S T, et al. (2001).
"dbSNP: the NCBI database of genetic variation". Nucleic Acids Res.
29 (1): 308-311; Kitts A, and Sherry S, (2009). The single
nucleotide polymorphism database (dbSNP) of nucleotide sequence
variation in The NCBI Handbook [Internet]. McEntyre J, Ostell J,
editors. Bethesda (Md.): National Center for Biotechnology
Information (US); 2002
(www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=handbook&part=ch5).
Multiple isoforms of certain proteins may exist, e.g., as a result
of alternative RNA splicing or editing. In general, where aspects
of this disclosure pertain to a gene or gene product, embodiments
pertaining to allelic variants or isoforms are encompassed unless
indicated otherwise. Certain embodiments may be directed to
particular sequence(s), e.g., particular allele(s) or
isoform(s).
[0317] Table 4 lists Gene ID and NCBI RefSeq accession numbers for
certain human Th17-associated cytokines and their receptors. It
will be appreciated that certain of the protein sequences are
precursor sequences. The mature form of the protein would lack a
secretion signal sequence present in the precursor. It will be
appreciated that the sequences described under the respective
accession numbers for the cytokines and cytokine receptors listed
in Table 4 are exemplary and that naturally occurring variants,
e.g., allelic variants, are encompassed in various embodiments.
Furthermore, it will be appreciated that for purposes of generating
a useful binding agent (e.g., an antibody) for use as a detection
reagent or therapeutic agent, variant sequences, short peptide
segments, etc., may be used in certain embodiments.
TABLE-US-00010 TABLE 4 Gene ID and Accession Numbers for Certain
Th17-Associated Cytokines and their Receptors Protein and mRNA mRNA
Protein Gene Official Alternate name Gene accession accession
Symbol/Name and comments ID number number IL23A/interleukin p19
51561 NM_016584 NP_057668 23, alpha subunit IL12B/interleukin p40;
IL23 beta 3593 NM_002187 NP_002178 12B subunit. IL23R/interleukin
alpha subunit 149233 NM_144701 NP_653302 23 receptor of the IL23
receptor IL12RB/interleukin beta subunit 3594 NM_005535 NP_005526
12 receptor, beta 1 of the IL23 (isoform 1) (isoform 1) receptor
NM_153701 NP_714912 (isoform 2) (isoform 2) IL17A 3605 NM_002190
NP_002181 IL17F 112744 NM_052872 NP_443104 IL-17RA/ 23765 NM_014339
NP_055154 interleukin 17 receptor A
[0318] In some embodiments an anti-Th17 agent is an anti-IL-23
agent. An IL-23 agent is an agent (e.g., a molecule or complex)
that partially or fully bocks, inhibits, neutralizes, prevents or
interferes with a biological activity of IL-23. In some embodiments
a biological activity of IL-23 is the ability to induce IL-17
production by activated T cells. IL-23 is a heterodimeric cytokine
composed of two subunits. The IL-23 beta subunit, also called p40,
is shared with another cytokine, interleukin-12 (IL-12). The IL-23
alpha subunit is also called p19. The IL-23 subunits are joined by
a disulfide bond. IL-23 signals via binding to a heterodimeric
receptor, composed of IL-12Rbetal (IL12RB1), which is shared by the
IL-12 receptor, and IL-23R (Parham C, et al. (2002) J. Immunol. 168
(11): 5699-708). IL-23R associates constitutively with Janus kinase
2 (JAK2), and also binds to transcription activator STAT3 in a
ligand-dependent manner. The IL-23 signal transduction cascade
parallels those of various other cytokines, in that ligand binding
leads to activation of JAKs. The JAKs then phosphorylate the IL-23R
at key sites, forming docking sites for the STATs. Subsequently,
the JAKs phosphorylate the STATs, which dimerize and translocate to
the nucleus where they activate target genes. In some embodiments
an anti-IL-23 agent comprises an antibody that binds to the p19 or
p40 subunit of IL-23. In some embodiments an anti-IL-23 agent,
e.g., an anti-IL-23 antibody, binds to the p40 subunit and inhibits
both IL-23 and IL-12.
[0319] Certain anti-IL-23 agents and methods of identifying and/or
making such agents are disclosed in U.S. Ser. No. 10/697,599. For
example, screening methods and assays that may be readily employed
by the ordinary skilled artisan to identify and/or produce a
variety of anti-IL-23 agents (referred to sometimes as "IL-23
antagonists" in U.S. Ser. No. 10/697,599) are disclosed.
[0320] In certain embodiments an anti-IL-23 antibody that binds to
the p40 subunit of IL-23 is ustekinumab or a fragment thereof.
Ustekinumab (experimental name CNTO 1275, proprietary commercial
name Stelara.RTM., Centocor; CAS Number: 815610-63-0) is a human
monoclonal antibody of the IgG1 subclass. Exemplary anti-IL-23
antibodies that bind to the p19 subunit of human IL-23, and
isolated nucleic acids that encode at least one anti-IL-23p19
antibody, vectors, host cells, and methods of making, are described
in U.S. Ser. No. 11/617,503. Additional anti-IL-23 antibodies that
bind to the p19 subunit are described in U.S. Ser. No.
11/762,738.
[0321] In some embodiments an anti-IL-23 agent comprises an
IL-23p40 specific immunoglobulin derived proteis (see, e.g., U.S.
Ser. No. 11/768,582).
[0322] In some embodiments an IL-23 inhibitor comprises a
polypeptide comprising a soluble IL-23R or a variant or fragment
thereof capable of binding to IL-23 in solution. In some
embodiments a soluble IL-23R lacks the portion of IL-23R encoded by
exon 9 of the IL-23R alpha gene. See, e.g., Yu, R Y, J Immunol.
(2010) 15; 185(12):7302-8. In some embodiments a soluble IL-23R
lacks the portion of IL-23 encoded by exon 9 and at least a portion
of exon 8 of the IL-23R alpha gene.
[0323] In some embodiments, IL-23 activity is inhibited by
interfering with IL-23 signal transduction, e.g., by inhibiting one
or more processes or proteins involved in the IL-23 signal
transduction pathway. For example, in some embodiments IL-23
signaling is inhibited using a JAK inhibitor or a STAT inhibitor.
In some embodiments a JAK inhibitor inhibits JAK expression.
Methods of use to inhibit JAK expression in some embodiments
include the use of RNAi agents (e.g., siRNA) or antisense
oligonucleotides. In some embodiments a JAK inhibitor inhibits JAK
binding to IL-23 receptor. In some embodiments a JAK inhibitor
inhibits JAK dimerization. In some embodiments a JAK inhibitor
inhibits JAK kinase activity. For example, in some embodiments a
JAK inhibitor binds to the JAK kinase domain, e.g., to the ATP
binding site. Numerous JAK inhibitors are known in the art. For
example, INCB028050 is an orally bioavailable JAK1/JAK2 inhibitor
with reported nanomolar potency against JAK1 (5.9 nM) and JAK2 (5.7
nM) (Fridman, J S, et al., J Immunol. 2010; 184(9):5298-307).
INCB028050 is reported to inhibit intracellular signaling of
multiple proinflammatory cytokines including IL-6 and IL-23 at
concentrations <50 nM. Small molecule JAK2 inhibitors include,
e.g., AZD1480 and AZ960.
[0324] In some embodiments a STAT inhibitor inhibits STAT
expression. Methods of use to inhibit STAT expression in some
embodiments include the use of RNAi agents (e.g., siRNA) or
antisense oligonucleotides. In some embodiments a STAT inhibitor
inhibits STAT binding to JAK. In some embodiments a STAT inhibitor
inhibits STAT dimerization or nuclear translocation. In some
embodiments a STAT inhibitor comprises a phosphopeptide which,
e.g., competes with STAT for binding to phosphorylated JAK.
WO/2008/151037 discloses certain peptide-based STAT inhibitors of
use in certain embodiments. In some embodiments a STAT inhibitor
inhibits STAT binding to DNA. For example, a decoy oligonucleotide
comprising a sequence substantially identical to an endogenous DNA
sequence to which STAT naturally binds in human cells may bind to
STAT and prevent it from binding to its endogenous binding site(s).
Small molecule STAT3 inhibitors include, e.g., STA-21, IS3 295, and
S3I-M2001. See Huang, S., Clin Cancer Res 2007; 13:1362-1366 and
references therein, which are incorporated herein by reference, for
further information regarding certain STAT inhibitors.
[0325] In some embodiments an anti-Th17 agent is an anti-IL-17
agent. An IL-17 agent is an agent (e.g., a molecule or complex)
that partially or fully bocks, inhibits, neutralizes, prevents or
interferes with a biological activity of IL-17. Exemplary
anti-IL-17 polypeptides, e.g., anti-IL-17 antibodies, are described
in, e.g., U.S. Ser. No. 11/658,344. Additional anti-IL-17
antibodies are described in U.S. Ser. No. 11/762,738. In some
embodiments an anti-IL-17 agent comprises at least a portion of an
IL-17 receptor, wherein the portion binds to IL-17. Exemplary IL-17
receptor polypeptides are disclosed in, e.g., U.S. Ser. No.
09/022,260.
[0326] It will be understood that a polypeptide comprising a
binding domain of any of the various anti-Th17 antibodies or other
polypeptides described herein can be transferred into other
polypeptide backbones or used as isolated agents in certain
embodiments. It will further be understood that variants may be
used. For example, a variant may be at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more identical to a binding domain
of a receptor. In some embodiments an antibody that competes with a
particular antibody known in the art for binding to a cytokine of
interest may be used. In some embodiments an antibody of the IgG
class is modified so that it lacks an Fc domain that may activate
complement. For example, a variable domain of an IgG1 antibody may
be grafted to a constant region of an IgG4 antibody.
VI. Pharmaceutical Compositions and Administration Approaches
[0327] Suitable preparations, e.g., substantially pure preparations
of a complement inhibitor may be combined with pharmaceutically
acceptable carriers or vehicles, etc., to produce an appropriate
pharmaceutical composition. The term "pharmaceutically acceptable
carrier or vehicle" refers to a non-toxic carrier or vehicle that
does not destroy the pharmacological activity of the compound with
which it is formulated. One of skill in the art will understand
that a carrier or vehicle is "non-toxic" if it is compatible with
administration to a subject in an amount appropriate to deliver the
compound without causing undue toxicity. Pharmaceutically
acceptable carriers or vehicles that may be used include, but are
not limited to, water, physiological saline, Ringer's solution,
sodium acetate or potassium acetate solution, 5% dextrose, and the
like. The composition may include other components as appropriate
for the formulation desired, e.g., as discussed herein.
Supplementary active compounds, e.g., compounds independently
useful for treating a subject suffering from a respiratory
disorder, can also be incorporated into the compositions. The
invention provides such pharmaceutical compositions comprising a
complement inhibitor and, optionally, a second active agent useful
for treating a subject suffering from a respiratory disorder.
[0328] In some embodiments, the invention provides a
pharmaceutically acceptable complement inhibitor or
pharmaceutically acceptable composition comprising a complement
inhibitor, packaged together with a package insert (label) approved
by a government agency responsible for regulating pharmaceutical
agents, e.g., the U.S. Food & Drug Administration. In some
embodiments, the invention provides a pharmaceutical pack
comprising: (a) a pharmaceutically acceptable complement inhibitor
in concentrated or solid form (e.g., as a lyophilized powder); (b)
a pharmaceutically acceptable carrier, diluent, or vehicle. In some
embodiments, a carrier, diluent, or vehicle is suitable for use to
deliver the composition using a nebulizer. In some embodiments, a
suitable carrier, diluent, or vehicle may be provided separately or
acquired by a health care provider from an appropriate source.
Optionally a pack contains instructions for dissolving or diluting
the complement inhibitor in the carrier, diluent, or vehicle to
produce a composition for administration. In some embodiments a
package insert states one or more indications that include one or
more chronic complement-mediated disorders, e.g., one or more
chronic respiratory disorders, e.g., asthma or COPD. In some
embodiments, the package insert states particular patient and/or
disease characteristics or criteria that define a patient
population or disease category for treatment of which the
composition has been approved for use. In some embodiments, the
package insert specifies that the composition may be or should be
administered according to a method of the present invention, e.g.,
according to a dosing schedule and/or using a dosing interval
described herein.
[0329] In general, a pharmaceutical composition can be administered
to a subject by any suitable route of administration including, but
not limited to, intravenous, intramuscular, subcutaneously, by the
respiratory route, etc. In some embodiments, local administration
to a tissue or organ affected by a complement-mediated disorder is
used. It will be understood that "administration" encompasses
directly administering a compound or composition to a subject,
instructing a third party to administer a compound or composition
to a subject, prescribing or suggesting a compound or composition
to a subject (e.g., for self-administration), self-administration,
and, as appropriate, other means of making a compound or
composition available to a subject. If administration is
accomplished using an implanted reservoir, administration can refer
to causing release of a composition or compound from the
reservoir.
[0330] Pharmaceutical compositions suitable for injectable use
(e.g., intravenous administration, subcutaneous or intramuscular
administration) typically include sterile aqueous solutions (where
water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersion. Sterile solutions can be prepared by incorporating the
compound in the required amount in an appropriate solvent,
optionally with one or a combination of ingredients such as buffers
such as acetates, citrates, lactates or phosphates; agents for the
adjustment of tonicity such as sodium chloride or dextrose;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid, glutathione, or sodium
bisulfate; chelating agents such as ethylenediaminetetraacetic
acid; and other suitable ingredients etc., as desired, followed by
filter-based sterilization. One of skill in the art will be aware
of numerous physiologically acceptable compounds that may be
included in a pharmaceutical composition. Other useful compounds
include, for example, carbohydrates, such as glucose, sucrose,
lactose; dextrans; amino acids such as glycine; polyols such as
mannitol. These compounds may, for example, serve as bulking agents
and/or stabilizers, e.g., in a powder and/or when part of the
manufacture or storage process involves lyophilization.
Surfactant(s) such as Tween-80, Pluronic-F108/F68, deoxycholic
acid, phosphatidylcholine, etc., may be included in a composition,
e.g., to increase solubility or to provide microemulsion to deliver
hydrophobic drugs. pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide, if desired. The parenteral
preparation can be enclosed in ampoules, disposable syringes or
infusion bags or multiple dose vials made of glass or plastic.
Preferably solutions for injection are sterile and acceptably free
of endotoxin.
[0331] Generally, dispersions are prepared by incorporating the
active compound into a sterile vehicle which contains a basic
dispersion medium and appropriate other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, methods of preparation
can include vacuum drying and freeze-drying which yields a powder
of the active ingredient plus any additional desired ingredient,
e.g., from a previously sterile-filtered solution thereof.
[0332] For administration by the respiratory route (inhalation), a
complement inhibitor may be delivered in the form of an aerosol
spray from a pressured container or dispenser which contains a
suitable propellant. A metered dose inhaler (MDI), dry powder
inhaler, or nebulizer may be used. The aerosol may comprise liquid
and/or dry particles (e.g., dry powders, large porous particles,
etc.). Suitable aqueous vehicles useful in various embodiments
include water or saline, optionally including an alcohol. In some
embodiments the composition comprises a surfactant suitable for
introduction into the lung. Other excipients suitable for pulmonary
administration can be used.
[0333] A variety of different devices are available for respiratory
administration. Nebulizers are devices that transform solutions or
suspensions of medications into aerosols that are suitable for
deposition in the lower airway. Nebulizer types include jet
nebulizers, ultrasonic wave nebulizers, and vibrating mesh
nebulizers. A partial list of available vibrating mesh nebulizers
includes eFlow (Pari), i-Neb (Respironics), MicroAir (Omron), IHSO
Nebulizer (Beurer), and Aeroneb.RTM. (Aerogen). A Respimat.RTM.
Soft Mist.TM. Inhaler (Boeringer Ingelheim) may be used. A metered
dose inhaler (MDI) is a handheld aerosol device that uses a
propellant to deliver the therapeutic agent. MDIs include a
pressurized metal canister that contains pharmacological agent in
suspension or solution, propellant, surfactant (typically), and
metering valve. Chloroflourocarbons (CFCs) had been widely used as
propellants but have been largely replaced by hydrofluorocarbons
(HFCs, also known as hydrofluoroalkanes (HFA)) such as HFC-134a and
HFC-227ea. Carbon dioxide and nitrogen are other alternatives. A
dry powder inhaler (DPI) is a breath-actuated device that delivers
the drug in the form of particles contained in a capsule or blister
that is punctured prior to use and typically does not employ a
propellant. Examples of DPIs currently used to deliver medications
for treating asthma and/or COPD include, e.g., Diskus, Aerolizer,
HandiHaler, Twisthaler, Flexhaler. Such devices may be used to
deliver a complement inhibitor in various embodiments of the
invention. Other exemplary DPI devices that may be used in various
embodiments include 3M.TM. Taper and 3M Conix.TM., TAIFUN.RTM.
(AKELA Pharma), Acu-Breathe.TM. (Respirics).
[0334] Inhalation accessory devices (IADs) generally fall into 2
categories: spacers and holding chambers. Spacers and holding
chambers extend the mouthpiece of the inhaler and direct the mist
of medication toward the mouth, reducing medication lost into the
air. Using a spacer with an MDI can help reduce the amount of drug
that sticks to the back of the throat, improving direction and
deposition of medication delivered by MDIs. Valved holding chambers
(VHCs) allow for a fine cloud of medication to stay in the spacer
until the patient breathes it in through a one-way valve, drawing
the dose of medicine into the lungs. Examples include Aerochamber
and Optichamber.
[0335] Particulate compositions may be characterized on the basis
of various parameters such as the fine particle fraction (FPF), the
emitted dose, the average particle density, and the mass median
aerodynamic diameter (MMAD). Suitable methods are known in the art,
some of which are described in U.S. Pat. Nos. 6,942,868 and
7,048,908 and U.S. Publication Nos. 20020146373, 20030012742, and
20040092470. In certain embodiments aerosol particles are between
approximately 0.5 .mu.m-10 .mu.m (MMAD), e.g., about 5 .mu.m for
respiratory delivery, though smaller or larger particles could also
be used. In certain embodiments particles having a mass mean
aerodynamic diameter of between 1 .mu.m and 25 .mu.m, e.g., between
1 .mu.m and 10 .mu.m, are used.
[0336] A dry particle composition containing particles smaller than
about 1 mm in diameter is also referred to herein as a dry powder.
A "dry" composition has a relatively low liquid content, so that
the particles are readily dispersible, e.g., in a dry powder
inhalation device to form an aerosol or spray. A "powder" consists
largely or essentially entirely of finely dispersed solid particles
that are relatively free flowing and capable of being readily
dispersed in an inhalation device and subsequently inhaled by a
subject, preferably so that a significant fraction of the particles
can reach a desired portion of the respiratory tract. In certain
embodiments large porous particles having mean geometric diameters
ranging between 3 and 15 .mu.m and tap density between 0.04 and 0.6
g/cm.sup.3 are used. See, e.g., U.S. Pat. No. 7,048,908; Edwards,
D. et al, Science 276:1868-1871, 1997; and Vanbever, R., et al.,
Pharmaceutical Res. 16:1735-1742, 1999).
[0337] Various considerations for respiratory delivery that may be
useful in embodiments of the present invention are discussed in
Bisgaard, H., et al., (eds.), Drug Delivery to the Lung, Vol. 26 in
"Lung Biology in Health and Disease", Marcel Dekker, New York,
2002. Aerosol devices are discussed, e.g., in Dolovich M B, Dhand
R. Lancet. (2011) 377(9770):1032-45.
[0338] Oral administration may be used in certain embodiments. Oral
compositions generally include an inert diluent or an edible
carrier. For the purpose of oral therapeutic administration, the
active compound can be incorporated with excipients and used in the
form of tablets, troches, or capsules, e.g., gelatin capsules.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring. A liquid composition can also be administered orally.
Formulations for oral delivery may incorporate agents to improve
stability within the gastrointestinal tract and/or to enhance
absorption.
[0339] For topical application, a complement inhibitor may be
formulated in a suitable ointment containing the active component
suspended or dissolved in one or more carriers. Carriers for
topical administration include, but are not limited to, mineral
oil, liquid petrolatum, white petrolatum, propylene glycol,
polyoxyethylene, polyoxypropylene compound, emulsifying wax and
water. Alternatively, the pharmaceutically acceptable compositions
can be formulated as a suitable lotion or cream containing a
compstatin analog suspended or dissolved in one or more
pharmaceutically acceptable carriers. Suitable carriers include,
but are not limited to, mineral oil, sorbitan monostearate,
polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol, and water.
[0340] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated may be used
in the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished, e.g., through the use of nasal
sprays or suppositories. In some embodiments, intranasal
administration is used, e.g., to administer a complement inhibitor
to a subject in need of treatment for nasal polyposis, chronic
rhinosinusitis, or allergic rhinitis. For transdermal
administration, the active compounds are typically formulated into
ointments, salves, gels, or creams as generally known in the
art.
[0341] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0342] Methods of local administration to the eye include, e.g.,
intraocular administration, e.g., intraocular injection, e.g.,
intravitreal injection. In some embodiments, administration is by
choroidal injection, transscleral injection, eyedrops or eye
ointments, transretinal, subconjunctival bulbar, intravitreal
injection, suprachoroidal injection, subtenon injection, scleral
pocket or scleral cutdown injection.
[0343] In certain embodiments of the invention, a complement
inhibitor is prepared with carrier(s) that will protect the
compound against rapid elimination from the body, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. For example, a compound may be
incorporated into or encapsulated in a microparticle or
nanoparticle formulation. Biodegradable, biocompatible polymers can
be used, such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, polyethers,
polylactic acid, PLGA, etc. Liposomes or other lipid-based
particles can be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No. 4,522,811
and/or other references listed herein. Depot formulations
containing a complement inhibitor may be used. The complement
inhibitor is released from the depot over time. One of ordinary
skill in the art will appreciate that the materials and methods
selected for preparation of a controlled release formulation,
implant, etc., should be such as to retain activity of the
compound. In some embodiments, a composition is free or essentially
free of one or more carrier(s) whose primary or only intended
purpose or effect would be to result in sustained or controlled
release of an active agent, e.g., a complement inhibitor.
[0344] In some embodiments, a complement inhibitor is used in
combination with one or more additional active agent(s) useful to
treat a disorder of interest herein (see, e.g., Brunton, L L, et
al. (eds.), Goodman and Gilman's The Pharmacological Basis of
Therapeutics, (e.g., 11th or 12th edition), McGraw-Hill, for
examples of such agents.) in some embodiments one or more
additional active agents is administered in the same composition as
a complement inhibitor. In some embodiments one or more additional
active agents is administered in a separate composition, which
separate composition may be administered prior to, at approximately
the same time as, or after administration of a complement
inhibitor. In some embodiments, use of a complement inhibitor
allows reduction in dose and/or frequency of administration of an
additional active agent while maintaining at least equivalent
disease control and/or benefit to the subject. It will be
understood that pharmaceutical compositions comprising an
additional active agent may be prepared using pharmaceutically
acceptable carriers and/or preparation methods described herein or
known in the art, and administered using routes of administration
described herein or known in the art.
[0345] In some embodiments a second active agent is an agent that
interferes with the DC-Th17-B-Ab-C-DC cycle by a mechanism distinct
from direct inhibition of a complement component or complement
activation. In some embodiments a second active agent may be an
anti-IL-23 agent or anti-IL-17 agent. In some embodiments a
pharmaceutical composition or pharmaceutical pack comprises a
second active agent that interferes with the DC-Th17-B-Ab-C-DC
cycle. In some embodiments a package insert specifies that two
agents are to be administered in combination. In some embodiments a
complement inhibitor, e.g., a compstatin analog, may be added to
any treatment regimen that comprises an anti-Th17 agent. In some
embodiments such addition permits a lower dose or increased dosing
interval of the anti-Th17 agent to be used, without reduction in
efficacy. In some embodiments such addition results in increased
efficacy.
[0346] When two or more therapies (e.g., compounds or compositions)
are used or administered "in combination" with each other, they may
be given at the same time, within overlapping time periods, or
sequentially (e.g., separated by up to 2-4 weeks in time), in
various embodiments of the invention. They may be administered via
the same route or different routes in various embodiments. They may
be administered in either order in various embodiments. In some
embodiments, the compounds or compositions are administered within
4, 8, 12, 24, 48, 72, or 96 hours of each other. In some
embodiments, a first agent is administered prior to or after
administration of the second agent, e.g., sufficiently close in
time that the two agents are present at useful levels within the
body at least once. In some embodiments, the agents are
administered sufficiently close together in time such that no more
than 90% of the earlier administered composition has been
metabolized to inactive metabolites or eliminated, e.g., excreted,
from the body, at the time the second compound or composition is
administered. In some embodiments, the agents are administered
sufficiently close together in time such that no more than 2 weeks
has elapsed since the earlier administered agent has been
metabolized to inactive metabolites or eliminated, e.g., excreted,
from the body, at the time the second agent is administered. In
some embodiments administration of two agents (e.g., a complement
inhibitor and a second agent that interferes with the
DC-Th17-B-Ab-C-DC cycle act additively, resulting in an effect that
would not be achieved by either agent alone. In some embodiments
administration of two agents (e.g., a complement inhibitor and a
second agent that interferes with the DC-Th17-B-Ab-C-DC cycle act
synergistically, resulting in an effect that is greater than an
additive effect and/or is qualitatively different to an additive
effect in a clinically and/or statistically significant way.
[0347] It will be appreciated that a complement inhibitor and/or
additional active agent(s) can be provided as a pharmaceutically
acceptable salt. Pharmaceutically acceptable salts include those
derived from pharmaceutically acceptable inorganic and organic
acids and bases. Examples of suitable acid salts include acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptanoate,
glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate,
pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,
pivalate, propionate, salicylate, succinate, sulfate, tartrate,
thiocyanate, tosylate and undecanoate. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts, if appropriate depending on the identity of the active
agent.
[0348] It will be understood that the pharmaceutically acceptable
carriers, compounds, and preparation methods mentioned herein are
exemplary and non-limiting. See, e.g., Remington: The Science and
Practice of Pharmacy. 21st Edition. Philadelphia, Pa. Lippincott
Williams & Wilkins, 2005, for additional discussion of
pharmaceutically acceptable compounds and methods of preparing
pharmaceutical compositions of various types.
[0349] A compound or composition, e.g., a pharmaceutical
composition, can be used or administered to a subject in an
effective amount. In some embodiments, an "effective amount" of an
active agent, e.g., a complement inhibitor, (or composition
containing an active agent) refers to an amount of the active agent
(or composition) sufficient to elicit one or more biological
response(s) of interest in, for example, a subject to whom the
active agent (or composition) is administered. As will be
appreciated by those of ordinary skill in the art, the absolute
amount of a particular agent that is effective may vary depending
on such factors as the biological endpoint, the particular active
agent, the target tissue, etc. Those of ordinary skill in the art
will further understand that an "effective amount" may be
administered in a single dose, or may be achieved by administration
of multiple doses. For example, in some embodiments, an effective
amount may be an amount sufficient to achieve one or more of the
following: (i) reduce the severity of one or more manifestations
(e.g., one or more symptoms or signs) of a chronic respiratory
disorder; (ii) cause a reduction in frequency and/or severity of
exacerbations (which reduction may result in, e.g., decreased days
of school or work lost, decreased physician and/or emergency room
visits, decreased hospitalization events, and/or decreased
mortality); (iii) permit a reduction in use of standard medication
for the disorder while maintaining at least equivalent disease
control; and/or (iv) inhibit or prevent a long-term pathological
change associated with the disorder; and/or (v) improve daily
function. In many embodiments, a therapeutically relevant effective
amount at least in part reduces one or more manifestations (e.g.,
symptoms) of a chronic disorder and/or returns one or more
physiological or biochemical parameters or indicators associated
with or causative of a chronic disorder at least partially to
normal. For example, in some embodiments, an effective amount may
be an amount sufficient to achieve one or more of the following:
(i) reduce the severity of one or more manifestations (e.g., one or
more symptoms or signs) of a chronic respiratory disorder; (ii)
reduce the magnitude of EAR, LAR, and/or DAR (as assessed, for
example, by maximum reduction in FEV.sub.1 and/or maximum reduction
in PEF measured within a relevant time period following an allergen
challenge); (iii) reduce likelihood of developing an EAR, LAR,
and/or DAR; (iv) cause a reduction in frequency and/or severity of
exacerbations (which reduction may result in, e.g., decreased days
of school or work lost, decreased physician and/or emergency room
visits, decreased hospitalization events, and/or decreased
mortality); (v) permit a reduction in use of ICS, OCS, leukotriene
modifiers, and/or Xolair while maintaining at least equivalent
disease control; (vi) inhibit or prevent airway remodeling; (vii)
improve daily function and/or exercise tolerance; and/or (viii)
reduce one or more indicators of airway inflammation. In many
embodiments in which an agent is administered to a subject in need
of treatment for a chronic respiratory disorder, a therapeutically
relevant effective amount at least in part reduces one or more
manifestations (e.g., symptoms) of a chronic respiratory disorder
and/or returns one or more physiological or biochemical parameters
or indicators associated with or causative of a chronic respiratory
disorder at least partially to normal.
[0350] Indicators of airway inflammation include, e.g., the
presence of increased numbers of inflammation-associated cells such
as white blood cells (e.g., eosinophils, lymphocytes, macrophages,
and/or neutrophils) and/or inflammatory mediators (e.g., chemokines
(e.g., eotaxin, thymus and activation-regulated chemokine (TARC),
macrophage-derived chemokine (MDC)), cytokines (e.g., TNFalpha,
IL-1beta, IL-4, IL-5, IL-13, IL-25), histamine, cysteinyl
leukotrienes, nitric oxide) in the airways, as compared with a
suitable reference level, e.g., a normal level. For example, the
number and/or concentration of cells and/or mediators may be above
the upper limit of the normal range in subjects not suffering from
a disorder (where "normal range" typically refers to a range of
within .+-.2 standard deviations from a mean value in a population
of subjects) or may be greater than a value (or average value)
measured in that subject when the subject's disorder is well
controlled. A reduction in symptom severity and/or frequency can be
statistically significant and/or clinically meaningful within the
sound judgment of a physician or other medical practitioner.
Determining whether a disorder is well controlled is within the
sound judgment of a physician or other medical practitioner.
Art-accepted guidelines may be used.
[0351] In some embodiments an effective amount results in reduction
of at least one parameter associated with Th17 cells and/or Th17
activity. In some embodiments an effective amount reduces the level
of at least one cytokine associated with Th17 cells and/or Th17
activity, e.g., a cytokine that promotes Th17 cell formation and/or
activity or a cytokine produced by Th17 cells. In some embodiments
a cytokine is IL-17, IL21, IL-22, or IL-23. In some embodiments an
effective amount results in a shift from Th17 to Treg cells. In
some embodiments a shift from Th17 cells to Treg cells is reflected
in an immune micro-environment that is relatively rich in IL-10 and
relatively poor in IL-17 and IL-23.
[0352] For treatment of AMD, an effective amount may be an amount
sufficient to achieve one or more of the following: (i) inhibit or
prevent drusen formation; (ii) cause a reduction in drusen number
and/or size (drusen regression); (iii) cause a reduction in or
prevent lipofuscin deposits; (iv) inhibit or prevent visual loss or
slow the rate of visual loss; (v) inhibit choroidal
neovascularization or slow the rate of choroidal
neovascularization; (vi) cause a reduction in size and/or number of
lesions characterized by choroidal neovascularization; (vii)
inhibit choroidal neovascularization or slow the rate of retinal
neovascularization; (viii) cause a reduction in size and/or number
of lesions characterized by retinal neovascularization; (ix)
improve visual acuity and/or contrast sensitivity; (x) inhibit or
prevent photoreceptor or RPE cell atrophy or apoptosis, or reduce
the rate of photoreceptor or RPE cell atrophy or apoptosis; (xi)
inhibit or prevent progression of non-exudative macular
degeneration to exudative macular degeneration; (xii) reduce one or
more indicia of inflammation, e.g., the presence of
inflammation-associated cells such as white blood cells (e.g.,
neutrophils, macrophages) in the eye, the presence of endogenous
inflammatory mediators, one or more symptoms such as eye pain,
redness, light sensitivity, blurred vision and floaters, etc.
[0353] One of skill in the art will be aware of appropriate methods
to assess the afore-mentioned biological effects and other
biological effects of interest. Symptoms can be assessed using
standardized instruments (e.g., questionnaires) known in the art.
Any of a variety of different health-related quality of life
(HRQOL) instruments can be used, which can be generic or
specifically associated with the respiratory system (e.g., asthma
and/or COPD-specific). Pulmonary function tests, particularly
spirometry, can be used to measure parameters of lung function that
are frequently altered in subjects with chronic respiratory
disorders, such as FEV.sub.1, FVC, FEV.sub.1/FVC, PEF, etc.
Allergen challenge can be performed, e.g., as described in Kelly M
M. J Allergy Clin Immunol. 125(2):349-356, 2010 or studies
described in Cockcroft, D W, et al. Can Respir J. 14(7):
414-418,2007. Myofibroblasts synthesize collagen and are believed
to play an important role in airway remodeling in disorders
characterized by chronic airway inflammation such as asthma and
COPD. These cells are increased in the airways of asthmatic
individuals 24 h after allergen challenge. Inhibition of the
increase in airway wall myofibroblasts that would otherwise occur
following allergen challenge may indicate decreased airway
remodeling potential. Alternately or additionally, features
associated with airway remodeling such as smooth muscle
hyperplasia, goblet cell hyperplasia, and/or subepithelial collagen
deposition can be assessed.
[0354] Bronchial hyperreactivity can be assessed using, for
example, "direct" and "indirect" challenge tests, which refer to
the mode of action of the agents in relation to smooth muscle
contraction. Methacholine chloride and histamine diphosphate are
most commonly used as direct smooth muscle stimuli. The most
frequently used indirect stimuli are hypertonic saline, adenosine
monophosphate (AMP), and mannitol. Challenge testing can be
performed, e.g., according to guidelines published by the ERS
(Sterk P J, et al. Airway responsiveness. Standardized challenge
testing with pharmacological, physical and sensitizing stimuli in
adults. Report Working Party Standardization of Lung Function
Tests, European Community for Steel and Coal. Official Statement of
the European Respiratory Society. Eur Respir J Suppl 1993;
16:53-83) and ATS (Crapo, R O, et al., Guidelines for methacholine
and exercise challenge testing-1999. Am J Respir Crit Care Med
2000; 161:309-329). Two suitable methods for inhaling aqueous
solutions of pharmacologic stimuli that may be used are the
2-minute tidal breathing method and the dosimeter method.
Bronchoconstriction causes increased airway resistance. PC(X)
(where X is a number, typically between 10 and 100) refers to the
amount of stimulus required to cause a decrease of X % in airway
resistance. In general, persons with bronchial hyperreactivity,
exhibit a decreased PC(X) than normal individuals. For example
individuals with bronchial hyperreactivity may have a methacholine
PC(20)<4 mg/ml, while individuals with bronchial hyperactivity
may have a PC(20) >4 mg/ml. In some embodiments, an effective
amount of a therapeutic agent increases PC(X) in subjects suffering
from a chronic respiratory disorder characterized by bronchial
hyperreactivity relative to control subjects.
[0355] Inflammation-associated cells and/or mediators may be
assessed, for example, in a suitable sample such as induced sputum,
BAL fluid, and/or airway tissue sample (e.g., obtained from a
biopsy such as an endobronchial biopsy). Cells, e.g.,
inflammation-associated cells can be detected and optionally
quantified using, e.g., electron microscopy, optical microscopy
(optionally using suitable chemical stains or antibodies to
particular markers (immunohistochemistry), flow cytometry, or other
suitable methods. Mediator (e.g., cytokine) levels may be measured
using, e.g., antibody-based assays such as ELISA assays, bead array
assays (such as the Luminex xMAP technology or Cytometric Bead
Array (CBA) system from BD Biosciences), antibody array assays, or
appropriate bioassays. Expression of mediators can alternately or
additionally be assessed by measuring the level of mRNA encoding
such mediators (e.g., using any suitable method for measuring RNA
level such as reverse transcription PCR, hybridization to
oligonucleotide or cDNA arrays, RNA-Seq (e.g., methods making use
of high-throughput sequencing technologies to sequence cDNA to
obtain information about RNA in a sample), etc.).
[0356] Exercise tolerance may be assessed, e.g., by testing
performance on a 6 minute walk test (e.g., wherein improved
exercise tolerance is evidenced by an increase in the distance a
subject is able to walk in 6 minutes), shuttle walk test, and/or
cardiopulmonary exercise testing. See, e.g., ATS Statement:
Guidelines for the Six-Minute Walk Test (2002) for discussion of 6
minute walk test.
[0357] In general, a control subject can be, e.g., an untreated
subject or a subject treated with a placebo. An "untreated subject"
may be a subject who has not received treatment with a complement
inhibitor within the preceding 6 months. In some embodiments, an
untreated subject has not received treatment with an ICS, OCS,
LTRA, and/or LABA within at least the preceding 4 weeks. In some
embodiments, an untreated subject has not received treatment with
an anti IgE agent within at least the preceding 12 weeks.
Historical control information can be used. In some embodiments, a
subject can serve as his or her own control. For example, one or
more parameters can be measured once or more prior to treatment and
once or more during and/or following treatment. In some
embodiments, an "active control" (or "active comparator") is used,
wherein a biological effect of the complement inhibitor is compared
with that of a compound known to affect the parameter being
assessed. For example, a compound that is approved for use as a
controller medication in asthma may be used. It will be appreciated
that if an active comparator is used as a control, an effective
amount of a complement inhibitor may have less, more, or about the
same effect as the active comparator at one or more time points in
various embodiments.
[0358] In some embodiments, one or more biological effect(s) of a
complement inhibitor is evident when tested at multiple time points
during a dosing interval of the instant invention, wherein said
time points encompass at least 75% of the dosing interval, e.g., at
least 80%, 85%, 90%, 95%, or more of the dosing interval. In some
embodiments, one or more biological effect(s) of a complement
inhibitor is evident when tested at or near the end of the dosing
interval, where "near the end of the dosing interval" means up to 2
days before the end of the dosing interval, e.g., on the day before
the end of the dosing interval.
[0359] In some embodiments, an animal model is used, for example,
to help guide selection of a dose, dose range, or formulation for
testing in human, to assess one or more biological effect(s), etc.
Commonly used animal models for airway inflammation and/or asthma
involve inhalation of Ascaris suum antigen. For example, inhalation
of Ascaris suum antigen by allergic monkeys (e.g., cynomolgus
monkey; Macaca fascicularis) causes an early bronchoconstriction
and delayed allergic reaction, including a pulmonary inflammatory
infiltrate. See, e.g., Mellado, M., et al., J Pharmacol Exp Ther.
(2008) 324(2):769-75; Zou, J., et al. Genome Biol. 2002;
3(5):research0020. Epub 2002 Apr. 11. Similar models exist in mice,
sheep, guinea pigs, etc. In some embodiments, a significant
reduction in allergen-induced EAR, LAR, and/or AHR (e.g., as
assessed using methacholine challenge) and/or a significant
increase in PC(X), in treated animals as compared with untreated
animals, indicates effectiveness. In some embodiments, a reduction
in EAR, LAR, and/or AHR remains evident at the end of a dosing
interval selected according to the instant invention (e.g.,
immediately prior to the next dose).
[0360] In general, appropriate doses of complement inhibitor or
other active agent depend at least in part upon the potency of the
complement inhibitor or other active agent, route of
administration, etc. In general, dose ranges that are effective and
well tolerated can be selected by one of ordinary skill in the art,
Such doses can be determined using clinical trials as known in the
art. Optionally, a dose may be tailored to the particular
recipient, for example, through administration of increasing doses
until a preselected desired response is achieved, such as a
preselected desired degree of complement inhibition and/or
preselected desired reduction in response to allergen challenge,
reduction in bronchial hyperreactivity, and/or reduction in one or
more symptoms of the disorder. If desired, the specific dose level
for any particular subject may be selected based at least in part
upon a variety of factors including the activity of the specific
compound employed, the particular condition being treated and/or
its severity, the age, body weight, general health, route of
administration, any concurrent medication, and/or the degree of
complement protein expression or activity measured in one or more
samples obtained from the subject. In some embodiments an effective
amount or dose ranges from about 0.001 to 500 mg/kg body weight,
e.g., about 0.01 to 100 mg/kg body weight, e.g., about 0.1 to 50
mg/kg body about 0.1 to 20 mg/kg body weight, e.g., about 1 to 10
mg/kg.
Example 1
Effect of a Potent Compstatin Analog in Ascaris suum Animal Model
of Asthma
[0361] A potent compstatin analog having the amino acid sequence of
the compstatin analog of SEQ ID NO: 28, was synthesized using
standard methods. Briefly, amino acids were obtained as
Fmoc-protected amino acids, in which the a-amino group of each
amino acid was protected with Fmoc. Side chain functional groups
were also blocked with various appropriate protective groups.
Synthesis was accomplished following the solid phase methodology
described by Merrifield (J. Amer. Chem. Soc. 85, 2149 (1963)).
Chain assembly was performed on solid phase, at the conclusion of
which the N-terminus was acetylated; the peptide was then cleaved
from the solid phase and simultaneously deprotected via acidolysis
using TFA and amidated. The linear peptide was then oxidized and
purified. A study designed to evaluate the efficacy of CA-28 after
14 days of administration in a non-human primate model of asthma
was performed. In this study, a dose of 15 mg/kg of CA-28 in a 2.0%
glycerol solution was administered to anesthetized animals
(cynomolgus monkeys) via intratracheal nebulization once a day for
14 consecutive days using a pneumatic nebulizer (Pari LC Plus, Pari
USA, Midlothian, Va.). Budesonide (10 mg/kg, administered once
daily for 9 days as a powder using an insufflator), a
glucocorticoid used for treatment of asthma, was used as a positive
control. Primary endpoints included the effects on bronchoalveolar
lavage (BAL) cell counts, cytokine levels, and acute pulmonary
function changes as assessed by airway resistance (RL) and dynamic
compliance (CDYN) after challenges with Ascaris suum (A. suum).
[0362] Animals were subjected to challenge with A. suum at 3 time
points (prior to initial dose--Challenge), on day 14 (Challenge 1,
i.e., the last day of dosing), and on day 30 (Challenge 2). While
each animal was anesthetized, a single dose of A. suum antigen was
administered via intermittent positive pressure breathing with a
ventilator and in-line nebulizer over 15 breaths. Each animal was
administered an optimum response dose (ORD) which is the dose of
antigen (dilution) that has historically elicited a >40%
increase in lung resistance (R.sub.L) and a >35% decrease in
dynamic compliance (C.sub.DYN). Blood was collected by venipuncture
and analyzed for routine clinical chemistry and hematology
parameters.
[0363] Broncheoalveolar lavage (BAL) was performed by guiding a
pediatric fiberoptic bronchoscope past the carina to wedge in a
major bronchus. An attempt was made to lavage different lung fields
at each time point. Three washes of sterile saline (20 mL each)
were instilled and immediately aspirated for collection into tubes.
The first wash collection was placed into one 50 mL conical tube
while the second and third wash collections were combined into a
second 50 mL conical tube. The samples were placed on wet ice or in
a refrigerator set to maintain 4.degree. C. until transport. The
cell pellets from the different wash combinations (1st/2nd/3rd
wash) were combined and analyzed for total and differential cell
counts. From stained slides, BAL cell morphology and differential
were determined by counting a minimum of 200 nucleated cells from
all washes (cell pellets were combined from all washes), if
available, if less than 200 nucleated cells were available this is
documented in the study records and results. Relative and absolute
counts were determined for macrophages, eosinophils, neutrophils,
lymphocytes, and mast cells. Erythrocytes, ciliated respiratory
cells, and squamous epithelial cells were not counted. BAL samples
were analyzed for eotaxin, RANTES, IL-4, IL-5, IL-6, IL-8, IL-10,
IL-13, IL-17a, IL-23, and INF-.gamma. using qualified methods.
[0364] Results
[0365] Following aerosol A. suum antigen challenge during Challenge
0 (control challenge prior to dosing), all animals exhibited a
severe bronchoconstrictor response, which was associated with
increases in lung resistance (RL) and decreases in dynamic
compliance (CDYN) followed by pulmonary eosinophilia.
[0366] CA-28 did not affect the acute phase bronchoconstriction
resulting from A. suum challenge at either Challenge 2 (on the last
day of CA-28 dosing) or Challenge 2 (28 days after the cessation of
dosing).
[0367] CA-28 resulted in slight, improvement (reduction) in
eosinophilia following Challenges 1 and 2. However, eosinophil
counts were higher in the baseline samples collected right after
dosing and prior to the first A. suum challenge.
[0368] Treatment with inhaled CA-28 at 15 mg/kg in a vehicle
comprised of 2.0% glycerol in water resulted in lower levels of
most upregulated cytokines and chemokines as compared to animals
treated with vehicle control in the treatment session, in a way
that was comparable to Budesonide in many case, most notably
eotaxin, IFN-.gamma., IL-4, IL-13, and IL-23, although the
suppression did not reach statistical significance in most cases,
due to the intrinsic high variability of the data and the low
number of animals. The most remarkable data was the total
suppression of IL-23 in CA-28 treated animals observed at all time
points following Challenges 1 and 2. Inhibition of most of the
other cytokines appeared to be present even following Challenge 2
in CA-28 treated groups. CA-28 upregulated baseline levels of
IL-10, a key regulatory cytokines, following both Challenges 1 and
2. Data are presented in graphical format in FIGS. 1-11.
[0369] The data are consistent with the conclusion that CA-28
creates a protective immune micro-environment (high IL-10, low
IFN.gamma./IL-4/IL-13/IL-17/IL-23) both when the drug is present in
the lung (Challenge 1) and 27 days following washout of the drug
(Challenge 2) (assuming a 1 day washout for both CA-28 and
Budesonide). In particular, IL-17 and 11-23 levels in CA-28 treated
animals 24 hours following Challenge 2 were lower than those in
control animals, suggesting a sustained beneficial effect. In the
case of Budesonide the IL-17/IL-23 axis appears to be upregulated
24 hours following Challenge 2.
[0370] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims. It will be appreciated that the invention is in no
way dependent upon particular results achieved in any specific
example or with any specific embodiment. Articles such as "a", "an"
and "the" may mean one or more than one unless indicated to the
contrary or otherwise evident from the context. Claims or
descriptions that include "or" between one or more members of a
group are considered satisfied if one, more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process unless indicated to the contrary or
otherwise evident from the context. The invention includes
embodiments in which exactly one member of the group is present in,
employed in, or otherwise relevant to a given product or process.
For example, and without limitation, it is understood that where
claims or description indicate that a residue at a particular
position may be selected from a particular group of amino acids or
amino acid analogs, the invention includes individual embodiments
in which the residue at that position is any of the listed amino
acids or amino acid analogs. The invention also includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process. Furthermore, it is to be understood that the invention
encompasses all variations, combinations, and permutations in which
one or more limitations, elements, clauses, descriptive terms,
etc., from one or more of the listed claims or from the description
above is introduced into another claim. For example, any claim that
is dependent on another claim can be modified to include one or
more elements, limitations, clauses, or descriptive terms, found in
any other claim that is dependent on the same base claim.
Furthermore, where the claims recite a composition, it is to be
understood that methods of administering the composition according
to any of the methods disclosed herein, and methods of using the
composition for any of the purposes disclosed herein are included
within the scope of the invention, and methods of making the
composition according to any of the methods of making disclosed
herein are included within the scope of the invention, unless
otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. Methods of treating a subject can include a step of
providing a subject in need of such treatment (e.g., a subject who
has had, or is at increased risk of having, a disease), a step of
diagnosing a subject as having a disease and/or a step of selecting
a subject for treatment with a complement inhibitor and/or
anti-Th17 agent. In some embodiments a method of treatment
comprises monitoring a subject for a Th17 biomarker. In some
embodiments a method of treatment comprises monitoring a subject
for a Th17 biomarker and retreating the subject based at least in
part on the result of such monitoring, e.g., administering a
complement inhibitor to the subject if the biomarker indicates a
resurgence of Th17 cells and/or Th17-associated activity.
[0371] Where elements are presented as lists, it is to be
understood that each subgroup of the elements is also disclosed,
and any element(s) can be removed from the group. For purposes of
conciseness only some of these embodiments have been specifically
recited herein, but the invention includes all such embodiments. It
should also be understood that, in general, where the invention, or
aspects or embodiments of the invention, is/are referred to as
comprising particular elements, features, etc., certain embodiments
of the invention or aspects of the invention consist, or consist
essentially of, such elements, features, etc.
[0372] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates otherwise.
Any embodiment, aspect, element, feature, etc., of the present
invention may be explicitly excluded from the claims. For example,
any complement inhibitor, anti-Th117 agent, carrier, formulation,
formulation component, disorder, subject population or
characteristic(s), dosing interval, administration route, or
combination thereof can be explicitly excluded.
Sequence CWU 1
1
72113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ile Cys Val Val Gln Asp Trp Gly His His Arg Cys
Thr 1 5 10 242PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 2Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Gln Asp Xaa Gly
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 35 40 35PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Xaa Gln Asp Xaa Gly 1 5
46PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Xaa Gln Asp Xaa Gly Xaa 1 5 513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Xaa
Xaa Xaa Xaa Gln Asp Xaa Gly Xaa Xaa Xaa Xaa Xaa 1 5 10
616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Xaa Xaa Cys Val Xaa Gln Asp Xaa Gly Xaa His Arg
Cys Xaa Xaa Xaa 1 5 10 15 716PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Xaa Xaa Cys Val Xaa Gln Asp
Xaa Gly Xaa His Arg Cys Xaa Xaa Xaa 1 5 10 15 813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Ile
Cys Val Val Gln Asp Trp Gly His His Arg Cys Thr 1 5 10
913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Ile Cys Val Val Gln Asp Trp Gly His His Arg Cys
Thr 1 5 10 1013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Ile Cys Val Tyr Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 1113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 1213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
1313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr 1 5 10 1413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 14Ile Cys Val Ala Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 1513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 15Ile Cys Val Ala Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 1613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Ile
Cys Val Ala Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
1713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Ile Cys Val Gly Gln Asp Trp Gly Ala His Arg Cys
Thr 1 5 10 1813PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 18Ile Cys Val Gly Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 1913PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 2013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Ile
Cys Val Phe Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
2113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Ile Cys Val Phe Gln Asp Trp Gly Ala His Arg Cys
Thr 1 5 10 2213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 22Ile Cys Val Xaa Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 2313PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 23Ile Cys Val Xaa Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 2413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Ile
Cys Val Trp Gln Asp Trp Gly Xaa His Arg Cys Thr 1 5 10
2516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Gly Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg
Cys Thr Ala Asn 1 5 10 15 2613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 26Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 2713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
2813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr 1 5 10 2913PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 29Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr 1 5 10 3013PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 3113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 31Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
3213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr 1 5 10 3315PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 33Gly Ile Cys Val Trp Gln Asp Trp Gly
Ala His Arg Cys Thr Asn 1 5 10 15 3413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
3513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr 1 5 10 3615PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 36Gly Ile Cys Val Trp Gln Asp Trp Gly
Ala His Arg Cys Thr Asn 1 5 10 15 3715PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Xaa
Xaa Xaa Xaa Xaa Gln Asp Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
3818PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Xaa Xaa Xaa Cys Val Xaa Gln Asp Xaa Gly Xaa His
Arg Cys Xaa Xaa 1 5 10 15 Xaa Xaa 3915PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
4015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Xaa Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg
Cys Ile Xaa 1 5 10 15 4115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 41Xaa Ile Cys Val Trp Gln Asp
Trp Gly Ala His Arg Cys Ile Xaa 1 5 10 15 4213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 42Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
4314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr Lys 1 5 10 4419PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 44Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Gly Gly Gly 1 5 10 15 Gly Gly Lys 4513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
4613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr 1 5 10 4714PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 47Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Lys 1 5 10 4813PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 4914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr Lys 1 5 10
5014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr Lys 1 5 10 5119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 51Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Gly Gly Gly 1 5 10 15 Gly Gly Lys 5219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Lys
Gly Gly Gly Gly Gly Ile Cys Val Trp Gln Asp Trp Gly Ala His 1 5 10
15 Arg Cys Thr 5314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 53Lys Ile Cys Val Trp Gln Asp Trp Gly
Ala His Arg Cys Thr 1 5 10 5414PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 54Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr Lys 1 5 10 5514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Ile
Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr Lys 1 5 10
5614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr Lys 1 5 10 5714PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 57Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Lys 1 5 10 5814PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 58Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr Lys 1 5 10 595PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 59Xaa
Pro Ala Trp Arg 1 5 605PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 60Lys Pro Ala Trp Arg 1 5
6110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Xaa Xaa Xaa Xaa Xaa Xaa Pro Ala Trp Arg 1 5 10
6210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Xaa Xaa Xaa Xaa Xaa Lys Pro Ala Trp Arg 1 5 10
636PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Phe Xaa Pro Ala Trp Arg 1 5 646PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 64Phe
Lys Pro Ala Trp Arg 1 5 655PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 65Xaa Pro Ala Trp Arg 1 5
665PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Xaa Pro Ala Trp Arg 1 5 676PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 67Phe
Xaa Pro Ala Trp Arg 1 5 6810PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 68Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn 1 5 10 6917PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 69Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 1 5 10 15 Thr 706PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
70His His His His His His 1 5 714PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 71Gln Asp Xaa Gly 1
7213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys
Thr 1 5 10
* * * * *
References